Novel chemistry used in biosensors

ABSTRACT

The invention relates to novel compositions of disubstituted bipyridyl osmium complexes useful for the synthesis of labeled proteins, nucleic acids, and for the modification of electrodes.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No. 13/187,142, filed Jul. 20, 2011, which claims the benefit of U.S. provisional application No. 61/366,013, filed Jul. 20, 2010 and is a continuation-in-part of U.S. patent application Ser. No. 12/253,875, filed Oct. 17, 2008 which in turn claims the benefit of priority to U.S. Provisional Patent Application Ser. Nos. 60/980,733, filed on Oct. 17, 2007, and 61/087,094 and 61/087,102, filed on Aug. 7, 2008, the entire contents and disclosures of each of which are herein incorporated by reference.

FIELD OF THE INVENTION

The invention relates to novel compositions and methods for the detection of analytes using change in E⁰ of target analytes.

BACKGROUND OF THE INVENTION

Electron transfer reactions are crucial steps in a wide variety of biological transformations ranging from photosynthesis or aerobic respiration. Studies of electron transfer reactions in both chemical and biological systems have led to the development of a large body of knowledge and a strong theoretical base, which describes the rate of electron transfer in terms of a small number of parameters.

Electronic tunneling in proteins and other biological molecules occurs in reactions where the electronic interaction of the redox centers is relatively weak. Semiclassical theory reaction predicts that the reaction rate for electron transfer depends on the driving force (−ΔG⁰), a nuclear reorganization parameter (λ), and the electronic-coupling strength between the reactants and products at the transition state (H_(AB)), according to the following equation:

k _(ET)=(4π³ /h ² λk _(B) T)^(1/2)(H _(AB))²exp[(−ΔG ⁰+λ)2/λk _(B) T]

The nuclear reorganization energy, λ, in the equation above is defined as the energy of the reactants at the equilibrium nuclear configuration of the products. For electron transfer reactions in polar solvents, the dominant contribution to λ arises from the reorientation of solvent molecules in response to the change in charge distribution of the reactants. The second component of λ comes from the changes in bond lengths and angles due to changes in the oxidation state of the donors and acceptors.

Previous work describes using change in reorganization energy, λ, as the basis of novel sensors. See for example, U.S. Pat. Nos. 6,013,459, 6,013,170, 6,248,229, and 7,267,939, all herein incorporated by reference in their entirety. The methods generally comprise binding an analyte to or near a redox active complex. The redox active complex comprises at least one electroactive molecule and a capture ligand which will bind the target analyte, and the complex is bound to an electrode. Upon analyte binding, the reorganization energy of the redox active molecule is altered, thus changing the E⁰, and allowing detection.

It is an object of the present invention to provide composition and methods for the detection of target analytes using alterations in the solvent reorganization energy, such as utilizing cyano ligands with the transition metals of the biosensor, corresponding to changes in the E⁰ of redox active molecules.

SUMMARY OF THE INVENTION

The present invention provides methods and compositions relating to biosensors for use in the detection of target analytes.

In one aspect, the invention provides compositions comprising a solid support (sometimes

referred to herein as a “substrate”) comprising an electrode comprising a covalently attached electroactive complex (EAM) with a particular E⁰. The substrates can optionally comprise an array of electrodes. The electrode(s) each comprise an EAM, that optionally can be part of a ReAMC. Suitable transition metals include iron, ruthenium and osmium, as well as others outlined herein. In some embodiments, the EAMs comprise at least one cyano ligand, with 2, 3, 4 and 5 also finding use in the invention. The EAMs (as well as the ReAMCs and diluent SAM forming species) can be linked to the electrodes using attachment linkers, including alkyl groups (including substituted alkyl groups).

In a further aspect, the electrodes optionally comprise self assembled monolayer (SAM) species.

In an additional aspect, the EAM/ReAMCs of the invention are attached to the electrode using an anchor ligand, which can be “unipodal” or “multipodal”, for example including the use of bipodal attachments such as two sulfur atoms or cyclic disulfide anchor groups.

In a further aspect, the EAM is part of a redox active capture complex (REAMC) comprising said EAM and a capture ligand. In one aspect, the capture ligand provides a coordination atom for the transition metal. In additional aspects, the capture ligand is separate from the EAM, such that the electrode comprises a first species comprising the EAM and a second species comprising a capture ligand.

In one aspect, the capture ligand is a protein, including peptides, or a carbohydrate.

In an additional aspect, the invention provides methods of detecting a target analytes comprising contacting a sample with a composition comprising an electrode as outlined herein. The binding of the target analyte to the capture ligand alters the E⁰ of the EAM, e.g. creating a second E⁰, which is measured to determine the presence or absence of the target analyte.

In a further aspect, the invention provides methods of making a biosensor comprising providing an electrode comprising a first species (usually a SAM forming species) comprising a first functional group. The electrode is contacted with a biomolecule (which will become the capture ligand) comprising a second functional group to form a covalent bond between the first species and the biomolecule. The electrode also comprises an electroactive complex (EAM), to form the biochips of the invention. In some aspects the functional groups on each molecule are selected from the group consisting of moieties comprising a maleimide, imidoester, N-hydroxysuccinimidyl, alkyl halide, aryl halide, alpha-haloacyl and pryidyl disulfide and cysteines (e.g. the first functional group comprises a maleimide and the biomolecule is a protein (e.g. peptide) comprising a cysteine amino acid.

In an additional aspect, the invention comprises compounds having the formula:

Anchor-Spacer 1-EAM-(Spacer 2)_(n)-CL  (I),

wherein said anchor comprises a cyclic-disulfide group,

EAM is an electroactive moiety comprises a solvent accessible redox compound,

CL is a capture ligand,

Spacer 1 is a SAM forming species, and

n=0 or 1.

In an additional aspect, the invention comprises compounds having the formula:

Anchor-Spacer 1-EAM-(Spacer 2)_(n)-CL  (I),

wherein EAM is an electroactive moiety comprising a transition metal and at least one charge-neutralizing ligand. The charge neutralizing ligand can be selected from the group consisting of: dithiocarbamate, benzenedithiolate, a Schiff base, EDTA, DTPA, carboxylate, amine, thiolate, phosphine, imidazole, pyridine, bipyridine, terpyridine, tacn, salen, acacen, Cp, pincer, scorpionates and pentaammine.

In one aspect, the invention provides compositions which are EAMs comprising osmium metal atoms having four cyano ligands and one bipyridyl ligand wherein the bipyridyl ligand is substituted independently with two moieties chosen from the group consisting of alkyl moieties and aminoalkyl moieties.

In a further aspect the EAMs have the chemical formula R¹,R²-bpy-Os(CN)₄ wherein bpy is bipyridyl and R¹ and R² are independently chosen from the group alkyl moiety and aminoalkyl moiety. The structural Formula (IV) of R¹,R²-bpy-Os(CN)₄ is as follows:

In a further embodiment of the invention the bipyridyl group of R¹,R²-bpy-Os(CN)₄ is either 4,4′ disubstituted (Formula IVa) or 5,5′ disubstituted (Formula IVb).

In yet another embodiment of the invention, R¹ and R² are independently moieties comprising substituted alkyl groups wherein the alkyls are substituted with moieties chosen from the group consisting of carboxylic acid, amine, maleimide, succinimide, N-hydroxysuccinimide, epoxide, and anhydride. As defined herein, the term “alkyl group” generally refers to linear or branched C₁-C₂₀, C₁-C₁₀, or C₁-C₆.

In a further embodiment of the invention, R¹ and R² are independently moieties comprising an electrophile and/or a nucleophile wherein the electrophile is chosen from the group consisting of activated ester, acyl azide, acyl halide, acyl nitrile, aldehydes, alkyl halide, alkyl halide, alkyl sulfonate, anhydride, aryl halide, aziridine, boronate, carboxylic acid, carbodiimide, diazoalkane, epoxide, haloacetamide, halotriazine, imido ester, isocyanate, isothiocyanate, ketone, maleimide, phosphoramidite, silyl halide, sulfonate ester, and sulfonyl halide and the nucleophile is chosen from the group consisting of amine, aniline, alcohol, phenol, hydrazine, hydroxylamine, carboxylic acid, thiol, and glycol.

In yet another embodiment of the invention, R¹ and R² are independently moieties comprising substituted alkyl groups wherein alkyls are substituted with moieties chosen from the group consisting of carboxylic acid, amine, maleimide, succinimide, N-hydroxysuccinimide, epoxide, and anhydride. As defined herein, the term “alkyl group” generally refers to linear or branched C₁-C₂₀, C₁-C₁₀, or C₁-C₆.

In yet another embodiment of the invention, R¹ and R² are independently substituted alkyl groups wherein the substituents are chosen from the group consisting of an electrophile and a nucleophile.

In another aspect, the invention provides compositions which are EAMs comprising a chemical compound having the chemical formula R¹,R²-bpy-Os(CN)₄ (See structural Formula IV) wherein R¹ and R² may be the same or different and are chosen from the group consisting of CH₃, CH₃(CH₂)_(m), and NH₂(CH₂)_(n) wherein m and n are independently chosen from the group consisting of 0, 1, 2, 3, 4, 5, and 6. The bpy may be chosen from the group consisting of 4,4′ substituted bpy (Formula IVa) and 5,5′ substituted bpy (Formula IVb).

In another aspect, the invention comprises representative compounds of the formula R¹,R²-bpy-Os(CN)₄ wherein R¹ and R² are as defined above selected from the group consisting of:

In another aspect. the invention provides a composition comprising a compound of formula R¹,R²-bpy-Os(CN)₄ wherein R¹ and R² are as defined above.

In another aspect, the invention provides compositions comprising an electrode comprising a covalently attached electroactive complex (EAM), said EAM comprising a compound of formula R¹,R²-bpy-Os(CN)₄ wherein R¹ and R² are as defined above.

In another aspect, the invention provides a method of detecting a target enzyme comprising:

a) contacting a sample with a composition comprising an electrode comprising:

-   -   i) an electroactive moiety (EAM) comprising a compound of of         formula R¹,R²-bpy-Os(CN)₄ wherein R¹ and R² are as defined         above.         with a first E⁰;     -   ii) a capture ligand;

-   under conditions whereby said target enzyme, if present, alters said     capture ligand such that said EAM has a second E⁰; and

b) measuring said second E⁰.

In one embodiment of the invention, the composition comprises a support comprising a plurality of electrodes each comprising:

i) an electroactive moiety (EAM) comprising a compound of formula R¹,R²-bpy-Os(CN)₄ wherein R¹ and R² are as defined above.

with a first E0;

ii) a capture ligand.

In another aspect, the invention provides a method of detecting a target enzyme comprising:

a) contacting a sample with a composition comprising a REAMC comprising:

-   -   i) an electroactive moiety (EAM) comprising a compound of         formula R¹,R²-bpy-Os(CN)₄ wherein R¹ and R² are as defined         above.         with a first E⁰;     -   ii) a capture ligand;

-   under conditions whereby said target enzyme, if present, alters said     capture ligand such that said EAM has a second E⁰; and

b) measuring said second E⁰.

In another aspect, the invention provides a composition comprising an electrode comprising:

-   -   a) a first species comprising a functional group; and     -   b) an electroactive complex (EAM), said EAM comprising a         compound of formula R¹,R²-bpy-Os(CN)₄ wherein R¹ and R² are as         defined above.

In one aspect, the functional group is a maleimide group.

In another aspect, the invention provides a method of making a biosensor comprising:

-   -   a) providing an electrode comprising:         -   i) a first species comprising a first functional group; and         -   ii) an electroactive complex (EAM), said EAM comprising a             compound of formula R¹,R²-bpy-Os(CN)₄ wherein R¹ and R² are             as defined above,     -   c) contacting said electrode with a biomolecule comprising a         second functional group to form a covalent bond between said         first species and said biomolecule.

In one embodiment of the invention, the first functional group is selected from the group consisting of moieties comprising a maleimide, imidoester, N-hydroxysuccinimidyl, alkyl halide, aryl halide, alpha-haloacyl and pryidyl disulfide.

In another embodiment of the invention, the first functional group comprises a maleimide and said biomolecule is a protein comprising a cysteine amino acid. The protein may be a peptide.

In another aspect, a compound is provided, the compound having the formula:

Anchor-Spacer 1-EAM-(Spacer 2)_(n)-CL  (I)

wherein said anchor comprises a cyclic-disulfide group, EAM is compound of formula R¹,R²-bpy-Os(CN)₄ wherein R¹ and R² are as defined above.

-   -   CL is a capture ligand,     -   Spacer 1 is a SAM forming species, and     -   n=0 or 1.

In one embodiment of the invention, the cyclic-disulfide group comprises pyridyl-oligophenylethylnyl-[1,2,5]-dithiazepane.

In another aspect, the invention provides a composition comprising an electrode comprising a self-assembled monolayer (SAM), wherein said SAM comprises a compound having the formula:

Anchor-Spacer 1-EAM-Spacer 2-CL  (II),

wherein said anchor is linked to said electrode group through a disulfide group,

-   -   EAM is a compound of formula R¹,R²-bpy-Os(CN)₄ wherein R¹ and R²         are as defined above,     -   CL is a capture ligand,     -   Spacer 1 is either insulating or conducting, and     -   Spacer 2 is optional.

In another aspect, the invention provides a method comprising:

a) providing a compound having the formula:

Anchor-Spacer 1-EAM-(Spacer 2)_(n)-CL  (II)

wherein said anchor comprises a cyclic-disulfide group,

EAM is a compound of formula R¹,R²-bpy-Os(CN)₄ wherein R¹ and R² are as defined above, CL is a capture ligand,

Spacer 1 is either insulating or conduction, and

-   -   n=0 or 1; and     -   b) contacting said compound to an electrode by opening said         cyclic disulfide to form an attachment of said anchor to said         electrode.

An one embodiment of the invention, an electrode further comprises a self-assembled monolayer (SAM).

In another aspect, the invention provides a method of detecting a target analyte in a test sample, comprising:

-   -   c) providing an electrode comprises a compound having the         formula:

Anchor-Spacer 1-EAM-(Spacer 2)_(n)-CL  (I),

wherein said anchor comprises a cyclic-disulfide group,

-   -   EAM is compound of formula R¹,R²-bpy-Os(CN)₄ wherein R¹ and R²         are as defined above,     -   CL is a capture ligand,     -   Spacer 1 is either insulating or conduction, and     -   n=0 or 1; and     -   d) contacting said electrode with said test sample; and     -   e) determining the presence of said target analyte by measuring         the reorganization energy of said EAM.

These and other aspects of the invention will be apparent in light of the description below.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1C depicts several compounds of the invention. FIG. 1A shows a compound comprising a capture ligand at one end that is linked to a redox here (here is shown to contain a Ruthenium example) through a spacer. The compound also comprises an anchor, through which the compound is attached to the surface of an electrode. Also shown are insulators (110) that are attached to the surface of the electrode as well. FIGS. 1B and 1C depicts a compound comprises multiple metals. The geometries such as the one shown, where CL is “capture ligand”, will attract a single protein and have it interact with two metal centers simultaneously giving a larger change in potential.

FIG. 2 depicts [BIM-Ru(NH3)4L]2+ complexes with BPAI anchors and [Ru(NH3)5L]2+ complexes with BPA anchors.

FIG. 3 depicts BIPOD based compounds.

FIG. 4A depicts [BIM-Ru(NH₃)₄L]²⁺ complexes with alkylthiol anchors. FIG. 4B depicts [Ru(NH₃)₅L]²⁺ complexes with conjugated thiol anchors.

FIG. 5A depicts new architectures for Ru—N based complexes. FIG. 5B depicts examples of Ru—N based complexes.

FIG. 6A schematically depicts modified Prussian blue surface for detection with amplification. FIG. 6B depicts the use of crown ether coordination to enhance potential shift.

FIG. 7 depicts the effect of second-sphere coordination, the adduct formation between Ru(NH₃)₅L and 18-C-6.

FIGS. 8A, 8B-1, and 8B-2 depict ligands used in the “single” and “side-by-side” arrangement when multiple metals are used.

FIGS. 9A-B depict some of the building blocks for generating the compound for detection of analyte.

FIGS. 10A and 10B depict some exemplary compounds.

FIG. 11 depicts some exemplary compounds.

FIGS. 12A-C and 13 depict several schematics of suitable geometries of the present invention. FIGS. 12A and C depict the situation where a linker is attached at one end to the electrode and the other end terminates in a ligand (L) that provides a coordination atom for the transition metal (TM). The capture substrate (CS) provides an additional ligand (not depicted), and a plurality of other ligands provide the remaining coordination atoms. Upon action by the enzyme, the capture substrate results in a leaving group (X). It should be noted that these Figures depicts a situation where the transition metal utilizes 6 coordination atoms, but other numbers of coordination atoms can be used, depending on the metal. Similarly, these Figures depicts the use of ligands that provide a single coordination atom, but fewer ligands providing multiple coordination atoms (e.g. multidentate) ligands can be used as well. FIG. 12B depicts the situation where the capture substrate and the EAM are attached separately to the electrode. FIG. 12C depicts a similar situation to FIG. 3A, except the capture substrate does not provide a coordination atom to the transition metal. It should be appreciated that solution phase systems can be similar to FIGS. 12 and 14, in that the electrochemical potential of the EAM in solution can be altered as a result of the enzymatic activity of the target enzyme.

FIG. 14 depicts a general scheme for producing the biochips of the invention.

FIG. 15 shows a specific example of the production of FIG. 14.

FIGS. 16A, B, C and D depict some exemplary compounds.

FIGS. 17A, B, C and D depict some exemplary compounds. Similar compounds can be constructed with different anchors, such as disulfide cyclic anchor groups, for example, or different spacers.

FIGS. 18A and B depict some exemplary compounds. Similar compounds can be constructed with different anchors, such as disulfide cyclic anchor groups, for example, or different spacers.

FIG. 19 depicts a general scheme of synthesis.

FIG. 20 depicts a general detection scheme.

FIGS. 21A, B, C, D and E depict some exemplary compounds. Similar compounds can be constructed with different anchors, such as disulfide cyclic anchor groups, for example, or different spacers.

FIG. 22 depicts a general scheme of synthesis.

FIG. 23 depicts a general scheme of synthesis for an assay, also described in the U.S. Ser. No. 12/253,875, filed Oct. 17, 2008, entitled “Novel Chemistry used in Biosensors”, hereby expressly incorporated by reference in its entirety.

FIG. 24 depict capture ligands. For the carboxylic acid reactant capture ligands: Anthrax Peptide is SEQ ID NO: 1 and SEB Peptide is SEQ ID NO: 2. For the maleimide reactive capture ligands: E. coli Peptide is SEQ ID NO: 3, PSA Peptide is SEQ ID NO: 4; SEB Peptide is SEQ ID NO: 5, and Anthrax Peptide is SEQ ID NO: 6.

FIGS. 25A, B, C and D depict some exemplary compounds. Similar compounds can be constructed with different anchors, such as disulfide cyclic anchor groups, for example, or different spacers.

FIGS. 26A, B, C and D depict some exemplary compounds using ferrocene as the EAM. Similar compounds can be constructed with different anchors, such as disulfide cyclic anchor groups, for example, or different spacers.

FIGS. 27 A and B depict some exemplary compounds. Similar compounds can be constructed with different anchors, such as disulfide cyclic anchor groups, for example, or different spacers.

FIG. 28 shows the electronic spectra for Compounds 301, 302, 303, 304, 305, and 306.

FIG. 29 shows the electronic spectra for Compounds 301, 302, 303, 304, 305, and 306 in water at pH 1.5 (gray line), pH 5.5 (thin black line) and pH 12 (thick black line).

FIG. 30 shows exemplary cyclic voltammograms for Compound 305 at pH 1.5 (top), pH 5.5 (center) and pH 12 (bottom).

DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed to improvements in electrochemical biosensors that rely on changes in the reorganization energy, λ, upon interaction of the target analyte and the biosensor, as evidenced by alterations in the observed E⁰. As shown previously, biosensors have been described that rely on changes in reorganization energy. The present invention has shown surprising improvements such as utilizing cyano ligands for the transition metal of the electroactive moieties (EAMs). The cyano ligands provide a surprising increase in the change of the E⁰; e.g., the delta in the E⁰ is higher than seen for other charged ligands.

I. Overview of Reorganization Enemy

The present invention provides methods and compositions for the detection of target analytes using changes in the reorganization energy of redox active molecules upon binding of the analytes, to facilitate or hinder electron transfer between the redox active molecule and an electrode. This invention is based on the fact that when a redox active molecule, such as a transition metal ion, is either oxidized (losing an electron) or reduced (gaining an electron), changes occur in the molecular structure as well as in its immediate solvent environment. These changes in the molecules structure (bond lengths and angles) and in the organization of the solvent molecules surrounding the molecule serve to stabilize the new oxidation state energetically. The sum of these changes constitute the reorganization energy, λ, of a redox reaction. The intramolecular changes are termed the inner-sphere reorganization energy, λ, and the changes in the solvent and environment are termed the outer-sphere or solvent reorganization energy, λ_(o).

For the purposes of this invention, the primary focus is on changes in the solvent reorganization energy although changes in the inner-sphere reorganization will also be considered in several embodiments of the invention. It is the intent of this invention to capitalize on changes in reorganization energy of a redox reaction when an electroactive molecule (EAM) is attached to a capture ligand (CL) which can selectively bind to an analyte of interest (e.g., protein or bacteria). Binding of the EAM-CL to the analyte results in a change in the solvent environment of the EAM so that the reorganization energy for a redox reaction involving the EAM is changed. For the case where the redox reaction involves electron transfer between an electrode and the EAM, the standard potential, E⁰, is changed. Thus, a change in E⁰ for an EAM-CL complex is an indication that it is bound to the analyte. It is the intent of this invention to detect the change in E⁰ as an indicator of binding and, consequently, the presence or absence of the analyte.

In conventional methodologies for analyte detection using electron transfer usually employ the EAM as a label or tag attached to one member of a binding pair (e.g., antibody and antigen). In these methods, EAM's are chosen in which the outer sphere solvent effect is minimal, by using electroactive molecules that have minimal solvent reorganization upon oxidation or reduction. Such EAMs generally comprise large hydrophobic ligands which have little interaction with water. Thus, the ligands for the transition metal ions traditionally used are non-polar and are generally hydrophobic, frequently containing organic rings (e.g., bipyridyl and terpyridyl). Such EAMs are chosen because conventionally because the magnitude of the total electron transfer reaction is measured (current) at a predetermined electrode potential.

Without being bound by theory, it is expected that the redox molecules best suited for this invention will be those whose redox reaction has a large solvent reorganization energy in aqueous environments. Solvent reorganization to stabilize an increase or decrease in charge can be attributed to several phenomena. In polar solvents such as water, the charge on a redox molecule is stabilized by orientation of the polar solvent molecules in the environment near the redox molecule. Since polar molecules have slight charge variation on different atoms of the molecule, their orientation around the redox molecule can help to stabilize it. Further, some ligands, such as CN⁻, themselves are polar and have partial charges on atoms. These polar ligands can themselves induce an orientation of surrounding solvent molecules. Stabilization (or destabilization) of charged redox molecules can also occur by hydrogen bonding of solvent and/or other molecules to the ligands of the transition metal in the redox molecule. Solvent molecules, as well as other molecules in the solvent surrounding a redox molecules can be characterized and compared based on their donor number or acceptor number (Neyhart et al., J. Am. Chem. Soc 118 (1996) 3724-29, incorporated herein by reference). The use of a particular solvent or a particular additive to a solvent of a molecule having a preferred donor or acceptor number would affect the solvent reorganization energy of a redox reaction. Further, a change in the charge of a redox molecule is stabilized by charged ion in the solvent. Thus, changes in solvent reorganization change upon analyte binding can be maximized by the proper choice of an electrolyte, considering the charge on the ions, the concentration of the ions, the size of the ions, and the hydrophobicity of the ions.

Without being bound by theory, it is preferred to maximize the stabilization of the redox molecule (i.e., maximize its solvent reorganization energy) in the solvent system of choice in order that the phenomena which stabilize the redox molecule are disrupted upon binding of the redox molecule/capture ligand complex, EAM-CL to the analyte. Under such conditions, one would expect that the change in reorganization energy, evidenced by a change in E⁰, would be optimum. It is expected that the binding of the CL to the analyte will “force” the EAM into an environment on the surface or in a cleft or pocket of the analyte (e.g., a protein) which will be less favorable to the optimal organization of the solvent environment. In one embodiment it is expected that binding would cause a shedding of water molecules near the EAM because of steric constraints.

It should be noted, and not being bound by theory, that whether the solvent reorganization energy increases or decreases upon binding (and whether E⁰ moves to more positive or to more negative potentials is dependent upon the particular charge of the EAM. If the EAM redox reaction being monitored results in an increased charge of the EAM, such as EAM²⁺ oxidation to EAM³⁺, then the bound environment of the EAM-CL would be less stabilized by reorganization than the unbound EAM-CL. Hence, one would expect the E⁰ to move to more positive potentials. Alternatively, if the EAM redox reaction being monitored results in a decreased charge of the EAM, such as EAM²⁻ oxidation to EAM⁻, then the unbound EAM-CL would be less stabilized by reorganization than the bound EAM-CL. Hence, one would expect the E⁰ to move to less positive potentials.

Without being bound by theory, there are two general mechanisms which may be exploited in the present invention. The first relates to inner sphere change due to the redox label. In this embodiment, the binding of a target analyte to a capture ligand which is sterically close to an EAM causes one or more of the small, polar ligands of the EAM to be replaced by one or more coordination atoms supplied by the target analyte, causing a change in the inner-sphere reorganization energy for at least two reasons. First, the exchange of a small, polar ligand for a putatively larger ligand will generally exclude more water from the metal, lowering the required solvent reorganization energy (i.e. an inner sphere λ_(i) effect). Secondly, the proximity of a generally large target analyte to the relatively small redox active molecule will sterically exclude water within the first or second coordination sphere of the metal ion, also changing the solvent reorganization energy.

Alternatively, the invention relies on substitutionally inert ligand, plus outer sphere effects. In this embodiment exchange of the polar ligands on the metal ion by a target analyte coordination atom. Rather, in this embodiment, the polar ligands are effectively irreversibly bound to the metal ion, and the change in solvent reorganization energy is obtained as a result of the exclusion of water in the first or second coordination sphere of the metal ion as a result of the binding of the target analyte; essentially the water is excluded (i.e. an outer sphere λ_(o) effect).

The present invention provides compounds with novel architecture and methods of using these compounds for detection of target analytes.

In some embodiments, the target analyte binds to the capture ligand. In some embodiments, the target analyte can be an enzyme, and the change in E⁰ is as a result of an enzymatic event, as described in U.S. Patent Application No. 61/087,094, hereby incorporated by reference in its entirety.

In the embodiments of the invention, there is a change in the E⁰, presumably due to a change in the reorganization energy, upon the introduction of the target analyte. As discussed more fully below, the change may be either a positive or negative shift in E0, depending on a variety of factors. In general, when cyano ligands are used, the change in E⁰ can be a negative shift in E⁰, although depending on the system and the other ligands used (if any), the effect of interaction of the target analyte with the capture ligand can result in a positive shift in E⁰. Surprisingly, shifts of greater than about 50 mV, 100 mV, 150 mV, 200 mV, 250 mV and 300 mV can be seen using cyano ligands.

CN— is a good nucleophile and in an example, the Iron-cyano (−2) complex has a partial charge on the N of a cyano of −0.8767 and has a partial charge on the C of a cyano of 0.5549. That charge number on the nitrogen is very large that one can consider it acting like there is a lone pair of electrons. Therefore, the partial negative charge on the N arranges water molecules with the protons of water oriented toward the N (the opposite is true for NH3 ligands). Since the partial charge on the nitrogen is high, this local orientation effect is strong and therefore the delta between water nearby (before target capture) and water excluded (after target capture) is high . . . i.e., the difference in lambda between the two states is quantitatively higher.

Unidentate CN— always binds through the carbon and therefore that large partial negative charge resides on the N. The larger that partial negative charge oriented toward the solvent (water) the larger the observable effect will be. As such, ligands with partial negative charges on the ligands stabilize high oxidation state metals, and have a strong impact on the orientation of water.

CN being the best because it has the highest net charge on the N and therefore the strongest interaction with protons of water:

CN⁻>NO₂ ⁻>SO₃ ²⁻>NCS⁻>NO

equivalent to NCS⁻ is SCN⁻.

In general, the more positive the Metal center becomes the higher the potential of the metal. Accordingly, when most if not all the negative charges are neutralized by interaction with water, the metal becomes more positive.

II. Geometries of the Sensors

The present invention is directed to methods and compositions for detection of target analytes, based on a change of electrochemical potential, E⁰, of a redox active molecule either on the surface of an electrode, or in some cases, in solution (while most of the description herein is directed to solid phase assays, as will be appreciated by those in the art, the invention can be used in solution as well, and such description herein is meant to apply as applicable to solution phase assays as well).

In general, the invention can be described as follows. A redox active molecule, generally comprising a transition metal and at least one ligand (such as one cyano ligand (or more, as described herein)) that provide coordination atoms for the transition metal, is attached to the surface of an electrode, generally through a linker as described herein. In addition, the electrode may also optionally comprise a self-assembled monolayer (SAM) as described herein. In the spatial vicinity of the redox active molecule, a capture ligand is also attached, generally in one of three ways, as described herein. Introduction and/or binding of the target analyte results in a change in the electrochemical potential of the redox active molecule, which is then detected in a variety of ways as described herein.

As depicted in FIGS. 12-14, there are three basic geometries for the sensor, although the descriptions herein are not meant to be so limited. In one embodiment, as shown in FIG. 12A, an electroactive moiety (EAM), comprising a transition metal ion and ligands that provide coordination atoms for the transition metal (in some embodiments, at least one of which is a cyano ligand), is attached to an electrode. In addition, a capture ligand (sometimes also referred to as a “binding ligand”) that will specifically bind the target analyte is also attached to the electrode. Both species are generally attached to the electrode using an attachment linker as described herein. The two species are attached to the electrode in such a manner that they are spatially close, such that the E⁰ of the EAM is altered upon binding of a target analyte. It should be noted that a third species, comprising a monolayer forming species, described below, can also be optionally present on the electrode. In this embodiment, the EAM species can have the formula (Ia), the capture ligand species can have the formula (Ib) and the diluent species can have the formula (Ic):

AG-Spacer 1-EAM  (Ia)

AG-Spacer 1-CL  (Ib)

AG-Spacer 1-TG,  (Ic)

wherein AG is an anchor group, EAM is an electroactive moiety comprises a solvent accessible redox complex, spacer 1 is a SAM forming species described herein, CL is a capture ligand, and TG is a terminal group, with n being 0 or 1.

In a second embodiment, one of the coordination atoms for the transition metal of the EAM is provided by the capture ligand, forming a “redox active moiety complex”, or ReAMC. In this embodiment, the coordination atom can be actually part of the capture ligand (e.g. if the capture ligand is a peptide, an amino group can provide the coordination atom) or part of a linker used to attach the capture ligand (e.g. a pyridine linker, etc.). The ReAMC is attached as a single species, and as above, an additional species, comprising a monolayer forming species, described below, can also be optionally present on the electrode. In this embodiment, the present invention provides a compound having the formula (II):

AG-Spacer 1-EAM-(Spacer 2)_(n)-CL  (II)

wherein AG is an anchor group, EAM is an electroactive moiety comprises a solvent accessible redox complex, CL is a capture ligand, spacer 1 is a SAM forming species described herein, and Spacer 2 is a linker, with n=0 or 1.

In a third embodiment, as depicted in FIG. 12C, there ReAMC is a single species, but the capture ligand does not provide a coordination atom; rather, it is spatially close but distinct from the EAM of the ReAMC. Again, a third species, comprising a monolayer forming species, described below, can also be optionally present on the electrode. In this embodiment, the present invention provides a compound having the formula (III):

wherein AG is an anchor group, EAM is an electroactive moiety comprises a solvent accessible redox complex, CL is a capture ligand, spacer 1 is a SAM forming species described herein, and S₂ and S₃ are two linkages that link the EAM and CL together with the AG to form a branched structure. S₂ and S₃ can be different or the same.

One example of this configuration is shown below:

where M=transitional metal; Ln=coordinating ligand that covalently connected to the anchor and capture ligand, n=0 or 1; and L=coordinating ligand.

III. Electrode

In one aspect, the present invention provides these ligand architectures attached to an electrode. By “electrode” herein is meant a composition, which, when connected to an electronic device, is able to sense a current or charge and convert it to a signal. Preferred electrodes are known in the art and include, but are not limited to, certain metals and their oxides, including gold; platinum; palladium; silicon; aluminum; metal oxide electrodes including platinum oxide, titanium oxide, tin oxide, indium tin oxide, palladium oxide, silicon oxide, aluminum oxide, molybdenum oxide (Mo2O6), tungsten oxide (WO3) and ruthenium oxides; and carbon (including glassy carbon electrodes, graphite and carbon paste). Preferred electrodes include gold, silicon, carbon and metal oxide electrodes, with gold being particularly preferred.

The electrodes described herein are depicted as a flat surface, which is only one of the possible conformations of the electrode and is for schematic purposes only. The conformation of the electrode will vary With the detection method used. For example, flat planar electrodes may be preferred for optical detection methods, or when arrays of nucleic acids are made, thus requiring addressable locations for both synthesis and detection. Alternatively, for single probe analysis, the electrode may be in the form of a tube, with the components of the system such as SAMs, EAMs and capture ligands bound to the inner surface. This allows a maximum of surface area containing the nucleic acids to be exposed to a small volume of sample.

The electrodes of the invention are generally incorporated into biochip cartridges and can take a wide variety of configurations, and can include working and reference electrodes, interconnects (including “through board” interconnects), and microfluidic components. See for example U.S. Pat. No. 7,312,087, incorporated herein by reference in its entirety.

The biochip cartridges include substrates comprising the arrays of biomolecules, and can be configured in a variety of ways. For example, the chips can include reaction chambers with inlet and outlet ports for the introduction and removal of reagents. In addition, the cartridges can include caps or lids that have microfluidic components, such that the sample can be introduced, reagents added, reactions done, and then the sample is added to the reaction chamber comprising the array for detection.

In a preferred embodiment, the biochips comprise substrates with a plurality of array locations. By “substrate” or “solid support” or other grammatical equivalents herein is meant any material that can be modified to contain discrete individual sites appropriate of the attachment or association of capture ligands. Suitable substrates include metal surfaces such as gold, electrodes as defined below, glass and modified or functionalized glass, fiberglass, Teflon, ceramics, mica, plastic (including acrylics, polystyrene and copolymers of styrene and other materials, polypropylene, polyethylene, polybutylene, polyimide, polycarbonate, polyurethanes, Teflon™, and derivatives thereof, etc.), GETEK (a blend of polypropylene oxide and fiberglass), etc., polysaccharides, nylon or nitrocellulose, resins, silica or silica-based materials including silicon and modified silicon, carbon, metals, inorganic glasses and a variety of other polymers, with printed circuit board (PCB) materials being particularly preferred.

The present system finds particular utility in array formats, i.e. wherein there is a matrix of addressable detection electrodes (herein generally referred to “pads”, “addresses” or “micro-locations”). By “array” herein is meant a plurality of capture ligands in an array format; the size of the array will depend on the composition and end use of the array. Arrays containing from about 2 different capture substrates to many thousands can be made.

In a preferred embodiment, the detection electrodes are formed on a substrate. In addition, the discussion herein is generally directed to the use of gold electrodes, but as will be appreciated by those in the art, other electrodes can be used as well. The substrate can comprise a wide variety of materials, as outlined herein and in the cited references.

In general, preferred materials include printed circuit board materials. Circuit board materials are those that comprise an insulating substrate that is coated with a conducting layer and processed using lithography techniques, particularly photolithography techniques, to form the patterns of electrodes and interconnects (sometimes referred to in the art as interconnections or leads). The insulating substrate is generally, but not always, a polymer. As is known in the art, one or a plurality of layers may be used, to make either “two dimensional” (e.g. all electrodes and interconnections in a plane) or “three dimensional” (wherein the electrodes are on one surface and the interconnects may go through the board to the other side or wherein electrodes are on a plurality of surfaces) boards. Three dimensional systems frequently rely on the use of drilling or etching, followed by electroplating with a metal such as copper, such that the “through board” interconnections are made. Circuit board materials are often provided with a foil already attached to the substrate, such as a copper foil, with additional copper added as needed (for example for interconnections), for example by electroplating. The copper surface may then need to be roughened, for example through etching, to allow attachment of the adhesion layer.

Accordingly, in a preferred embodiment, the present invention provides biochips (sometimes referred to herein “chips”) that comprise substrates comprising a plurality of electrodes, preferably gold electrodes. The number of electrodes is as outlined for arrays. Each electrode preferably comprises a self-assembled monolayer as outlined herein. In a preferred embodiment, one of the monolayer-forming species comprises a capture ligand as outlined herein. In addition, each electrode has an interconnection, that is attached to the electrode at one end and is ultimately attached to a device that can control the electrode. That is, each electrode is independently addressable.

Finally, the compositions of the invention can include a wide variety of additional components, including microfluidic components and robotic components (see for example U.S. Pat. Nos. 6,942,771 and 7,312,087 and related cases, both of which are hereby incorporated by reference in its entirety), and detection systems including computers utilizing signal processing techniques (see for example U.S. Pat. No. 6,740,518, hereby incorporated by reference in its entirety).

A. Self Assembled Monolayer Spacers

In some embodiments, the electrodes optionally further comprise a SAM. By “monolayer” or “self-assembled monolayer” or “SAM” herein is meant a relatively ordered assembly of molecules spontaneously chemisorbed on a surface, in which the molecules are oriented approximately parallel to each other and roughly perpendicular to the surface. Each of the molecules includes a functional group that adheres to the surface, and a portion that interacts with neighboring molecules in the monolayer to form the relatively ordered array. A “mixed” monolayer comprises a heterogeneous monolayer, that is, where at least two different molecules make up the monolayer. As outlined herein, the use of a monolayer reduces the amount of non-specific binding of biomolecules to the surface, and, in the case of nucleic acids, increases the efficiency of oligonucleotide hybridization as a result of the distance of the oligonucleotide from the electrode. Thus, a monolayer facilitates the maintenance of the target enzyme away from the electrode surface. In addition, a monolayer serves to keep charge carriers away from the surface of the electrode. Thus, this layer helps to prevent electrical contact between the electrodes and the ReAMs, or between the electrode and charged species within the solvent. Such contact can result in a direct “short circuit” or an indirect short circuit via charged species which may be present in the sample. Accordingly, the monolayer is preferably tightly packed in a uniform layer on the electrode surface, such that a minimum of “holes” exist. The monolayer thus serves as a physical barrier to block solvent accessibility to the electrode.

In some embodiments, the monolayer comprises conductive oligomers. By “conductive oligomer” herein is meant a substantially conducting oligomer, preferably linear, some embodiments of which are referred to in the literature as “molecular wires”. By “substantially conducting” herein is meant that the oligomer is capable of transferring electrons at 100 Hz. Generally, the conductive oligomer has substantially overlapping Tr-orbitals, i.e. conjugated Tr-orbitals, as between the monomeric units of the conductive oligomer, although the conductive oligomer may also contain one or more sigma (σ) bonds. Additionally, a conductive oligomer may be defined functionally by its ability to inject or receive electrons into or from an associated EAM. Furthermore, the conductive oligomer is more conductive than the insulators as defined herein. Additionally, the conductive oligomers of the invention are to be distinguished from electroactive polymers, that themselves may donate or accept electrons.

A more detailed description of conductive oligomers is found in WO/1999/57317, herein incorporated by reference in its entirety. In particular, the conductive oligomers as shown in Structures 1 to 9 on page 14 to 21 of WO/1999/57317 find use in the present invention. In some embodiments, the conductive oligomer has the following structure:

In addition, the terminus of at least some of the conductive oligomers in the monolayer is electronically exposed. By “electronically exposed” herein is meant that upon the placement of an EAM in close proximity to the terminus, and after initiation with the appropriate signal, a signal dependent on the presence of the EAM may be detected. The conductive oligomers may or may not have terminal groups. Thus, in a preferred embodiment, there is no additional terminal group, and the conductive oligomer terminates with a terminal group; for example, such as an acetylene bond. Alternatively, in some embodiments, a terminal group is added, sometimes depicted herein as “0”. A terminal group may be used for several reasons; for example, to contribute to the electronic availability of the conductive oligomer for detection of EAMs, or to alter the surface of the SAM for other reasons, for example to prevent non-specific binding. For example, there may be negatively charged groups on the terminus to form a negatively charged surface such that when the target analyte is nucleic acid such as DNA or RNA, the nucleic acid is repelled or prevented from lying down on the surface, to facilitate hybridization. Preferred terminal groups include —NH, —OH, —COOH, and alkyl groups such as —CH₃, and (poly)alkyloxides such as (poly)ethylene glycol, with —OCH₂CH₂OH, —(OCH₂CH₂O)₂H, —(OCH₂CH₂O)₃H, and —(OCH₂CH₂O)₄H being preferred.

In one embodiment, it is possible to use mixtures of conductive oligomers with different types of terminal groups. Thus, for example, some of the terminal groups may facilitate detection, and some may prevent non-specific binding.

In some embodiments, the electrode further comprises a passivation agent, preferably in the form of a monolayer on the electrode surface. For some analytes the efficiency of analyte binding (i.e. hybridization) may increase when the binding ligand is at a distance from the electrode. In addition, the presence of a monolayer can decrease non-specific binding to the surface (which can be further facilitated by the use of a terminal group, outlined herein. A passivation agent layer facilitates the maintenance of the binding ligand and/or analyte away from the electrode surface. In addition, a passivation agent serves to keep charge carriers away from the surface of the electrode. Thus, this layer helps to prevent electrical contact between the electrodes and the electron transfer moieties, or between the electrode and charged species within the solvent. Such contact can result in a direct “short circuit” or an indirect short circuit via charged species which may be present in the sample. Accordingly, the monolayer of passivation agents is preferably tightly packed in a uniform layer on the electrode surface, such that a minimum of “holes” exist. Alternatively, the passivation agent may not be in the form of a monolayer, but may be present to help the packing of the conductive oligomers or other characteristics.

The passivation agents thus serve as a physical barrier to block solvent accessibility to the electrode. As such, the passivation agents themselves may in fact be either (1) conducting or (2) nonconducting, i.e. insulating, molecules. Thus, in one embodiment, the passivation agents are conductive oligomers, as described herein, with or without a terminal group to block or decrease the transfer of charge to the electrode. Other passivation agents which may be conductive include oligomers of —(CF₂)_(n)—, —(CHF)_(n)— and —(CFR)_(n)—. In a preferred embodiment, the passivation agents are insulator moieties.

In some embodiments, the monolayers comprise insulators. An “insulator” is a substantially nonconducting oligomer, preferably linear. By “substantially nonconducting” herein is meant that the rate of electron transfer through the insulator is slower than the rate of electron transfer through the conductive oligomer. Stated differently, the electrical resistance of the insulator is higher than the electrical resistance of the conductive oligomer. It should be noted however that even oligomers generally considered to be insulators, such as —(CH2)16 molecules, still may transfer electrons, albeit at a slow rate.

In some embodiments, the insulators have a conductivity, S, of about 10-7 Ω-1 cm-1 or lower, with less than about 10-8 Ω-1 cm-1 being preferred. Gardner et al., Sensors and Actuators A 51 (1995) 57-66, incorporated herein by reference.

Generally, insulators are alkyl or heteroalkyl oligomers or moieties with sigma bonds, although any particular insulator molecule may contain aromatic groups or one or more conjugated bonds. By “heteroalkyl” herein is meant an alkyl group that has at least one heteroatom, i.e. nitrogen, oxygen, sulfur, phosphorus, silicon or boron included in the chain. Alternatively, the insulator may be quite similar to a conductive oligomer with the addition of one or more heteroatoms or bonds that serve to inhibit or slow, preferably substantially, electron transfer. Preferably, the alkyl or heteroalkyl chains are from about four to about 18 atoms in length, and more preferably from about six to about 16 atoms in length/

The passivation agents, including insulators, may be substituted with R groups as defined herein to alter the packing of the moieties or conductive oligomers on an electrode, the hydrophilicity or hydrophobicity of the insulator, and the flexibility, i.e. the rotational, torsional or longitudinal flexibility of the insulator. For example, branched alkyl groups may be used. In addition, the terminus of the passivation agent, including insulators, may contain an additional group to influence the exposed surface of the monolayer, sometimes referred to herein as a terminal group (“TG”). For example, the addition of charged, neutral or hydrophobic groups may be done to inhibit non-specific binding from the sample, or to influence the kinetics of binding of the analyte, etc. For example, there may be charged groups on the terminus to form a charged surface to encourage or discourage binding of certain target analytes or to repel or prevent from lying down on the surface.

The length of the passivation agent will vary as needed. Generally, the length of the passivation agents is similar to the length of the conductive oligomers, as outlined above. In addition, the conductive oligomers may be basically the same length as the passivation agents or longer than them, resulting in the binding ligands being more accessible to the solvent.

The monolayer may comprise a single type of passivation agent, including insulators, or different types. Suitable insulators are known in the art, and include, but are not limited to, —(CH₂)_(n)—, —(CRH)_(n)—, and —(CR₂)_(n)—, ethylene glycol or derivatives using other heteroatoms in place of oxygen, i.e. nitrogen or sulfur (sulfur derivatives are not preferred when the electrode is gold). Preferably, insulators are of the form —(CH²)_(n)— having a thiol or disulfide terminus for attachment to gold. Also preferable, the alternate end of the insulator is terminated in a hydrophilic group such as oligoethylene glycol, —OH, or —COOH.

In some embodiments, the electrode is a metal surface and need not necessarily have interconnects or the ability to do electrochemistry.

B. Anchor Groups

The present invention provides compounds comprising an anchor group. By “anchor” or “anchor group” herein is meant a chemical group that attaches the compounds of the invention to an electrode.

As will be appreciated by those in the art, the composition of the anchor group will vary depending on the composition of the surface to which it is attached. In the case of gold electrodes, both pyridinyl anchor groups and thiol based anchor groups find particular use.

The covalent attachment of the conductive oligomer may be accomplished in a variety of ways, depending on the electrode and the conductive oligomer used. Generally, some type of linker is used, as depicted below as “A” in Structure 1, where X is the conductive oligomer, and the hatched surface is the electrode:

In this embodiment, A is a linker or atom. The choice of “A” will depend in part on the characteristics of the electrode. Thus, for example, A may be a sulfur moiety when a gold electrode is used. Alternatively, when metal oxide electrodes are used, A may be a silicon (silane) moiety attached to the oxygen of the oxide (see for example Chen et al., Langmuir 10:3332-3337 (1994); Lenhard et al., J. Electroanal. Chem. 78:195-201 (1977), both of which are expressly incorporated by reference). When carbon based electrodes are used, A may be an amino moiety (preferably a primary amine; see for example Deinhammer et al., Langmuir 10:1306-1313 (1994)). Thus, preferred A moieties include, but are not limited to, silane moieties, sulfur moieties (including alkyl sulfur moieties), and amino moieties.

In some embodiments, the electrode is a carbon electrode, i.e. a glassy carbon electrode, and attachment is via a nitrogen of an amine group. A representative structure is depicted in Structure 15 of US Patent Application Publication No. 20080248592, hereby incorporated by reference in its entirety but particularly for Structures as described therein and the description of different anchor groups and the accompanying text. Again, additional atoms may be present, i.e. linkers and/or terminal groups.

In Structure 16 of US Patent Application Publication No. 20080248592, hereby incorporated by reference as above, the oxygen atom is from the oxide of the metal oxide electrode. The Si atom may also contain other atoms, i.e. be a silicon moiety containing substitution groups. Other attachments for SAMs to other electrodes are known in the art; see for example Napier et al., Langmuir, 1997, for attachment to indium tin oxide electrodes, and also the chemisorption of phosphates to an indium tin oxide electrode (talk by H. Holden Thorpe, CHI conference, May 4-5, 1998).

In one preferred embodiment, indium-tin-oxide (ITO) is used as the electrode, and the anchor groups are phosphonate-containing species.

1). Pyridinyl Anchor Groups

In one aspect, the present invention provides the use of pyridine and derivatives thereof to attach the compounds of the invention to the surface.

In some embodiments, the anchor comprises a pyridyl group, having the structure of formula (ID:

where the carbons on the ring can optionally and independently be substituted, using R groups as defined herein. Pyridine is a heterocyclic aromatic organic compound that is structurally related to benzene, wherein one CH group in the six-membered ring is replaced by a nitrogen atom. Pyridine can be used as a ligand in coordination chemistry. As a ligand, it is usually abbreviated as “py.” The invention utilizes the ability of the lone electron pair on the nitrogen atom of the pyridine to bind to metal surfaces. One advantage of the pyridine based compounds is that they are air stable. Curtis et al., Inorg. Chem. 24:385-397 (1985); Callahan et al., Inorg. Chem. 14:1443-1453 (1975); Lavallee and Fleischer, J. Am. Chem. Soc. 94:2583-2599 (1972); and Jwo et al., J. Am. Chem. Soc. 101:6189-6197 (1979), all of which are incorporated by reference.

In some embodiments, the pyridyl group comprises a bipyridyl group (Bispyridylacetylene, BPA), comprising two pyridyl groups separated by an acetylene group, shown below:

In this embodiment, the carbons on either ring can be optionally and independently be substituted, using R groups as defined herein. One of the rings will contain a linkage to a spacer, as defined herein, or, as shown in some of the figures, there may be more than one spacer attached to the pyridyl group (e.g. n=1 or more, with 2 finding particular use in some embodiments).

2). Sulfur Anchor Groups

Although depicted in Structure 1 as a single moiety, the conductive oligomer may be attached to the electrode with more than one “A” moiety; the “A” moieties may be the same or different. Thus, for example, when the electrode is a gold electrode, and “A” is a sulfur atom or moiety, multiple sulfur atoms may be used to attach the conductive oligomer to the electrode, such as is generally depicted below in Structures 2, 3 and 4. As will be appreciated by those in the art, other such structures can be made. In Structures 2, 3 and 4 the A moiety is just a sulfur atom, but substituted sulfur moieties may also be used.

Thus, for example, when the electrode is a gold electrode, and “A” is a sulfur atom or moiety, such as generally depicted below in Structure 6, multiple sulfur atoms may be used to attach the conductive oligomer to the electrode, such as is generally depicted below in Structures 2, 3 and 4. As will be appreciated by those in the art, other such structures can be made. In Structures 2, 3 and 4, the A moiety is just a sulfur atom, but substituted sulfur moieties may also be used.

It should also be noted that similar to Structure 4, it may be possible to have a conductive oligomer terminating in a single carbon atom with three sulfur moieties attached to the electrode.

In another aspect, the present invention provide anchor comprise conjugated thiols.

Some exemplary complexes with conjugated thiol anchors are shown in FIG. 4. In some embodiments, the anchor comprises an alkylthiol group. Some of the examples are shown in FIGS. 4A and 4B. The two compounds depicts in FIG. 4B are based on carbene and 4-pyridylalanine, respectively.

In another aspect, the present invention provides conjugated multipodal thio-containing compounds that serve as anchoring groups in the construction of electroactive moieties for analyte detection on electrodes, such as gold electrodes. That is, spacer groups (which can be attached to EAMs, ReAMCs, or an “empty” monolayer forming species) are attached using two or more sulfur atoms. These multipodal anchor groups can be linear or cyclic, as described herein.

In some embodiments, the anchor groups are “bipedal”, containing two sulfur atoms that will attach to the gold surface, and linear, although in some cases it can be possible to include systems with other multipodalities (e.g. “tripodal”). Such a multipodal anchoring group display increased stability and/or allow a greater footprint for preparing SAMs from thiol-containing anchors with sterically demanding head groups.

In some embodiments, the anchor comprises cyclic disulfides (“bipod”). Although in some cases it can be possible to include ring system anchor groups with other multipodalities (e.g. “tripodal”). The number of the atoms of the ring can vary, for example from 5 to 10, and also includes multicyclic anchor groups, as discussed below

In some embodiments, the anchor groups comprise a [1,2,5]-dithiazepane unit which is seven-membered ring with an apex nitrogen atom and a intramolecular disulfide bond as shown below:

In Structure (IIIa), it should also be noted that the carbon atoms of the ring can additionally be substituted. As will be appreciated by those in the art, other membered rings are also included. In addition, multicyclic ring structures can be used, which can include cyclic heteroalkanes such as the [1,2,5]-dithiazepane shown above substituted with other cyclic alkanes (including cyclic heteroalkanes) or aromatic ring structures.

In some embodiments, the anchor group and part of the spacer has the structure shown below

The “R” group herein can be any substitution group, including a conjugated oligophenylethynylene unit with terminal coordinating ligand for the transition metal component of the EAM.

The anchors are synthesized from a bipodal intermediate (I) (the compound as formula III where R=I), which is described in Li et al., Org. Lett. 4:3631-3634 (2002), herein incorporated by reference. See also Wei et al, J. Org, Chem. 69:1461-1469 (2004), herein incorporated by reference.

The number of sulfur atoms can vary as outlined herein, with particular embodiments utilizing one, two, and three per spacer.

C. Electroactive Moieties

In addition to anchor groups, the present invention provides compound comprising electroactive moieties. By “electroactive moiety (EAM)” or “transition metal complex” or “redox active molecule” or “electron transfer moiety (ETM)” herein is meant a metal-containing compound which is capable of reversibly or semi-reversibly transferring one or more electrons. It is to be understood that electron donor and acceptor capabilities are relative; that is, a molecule which can lose an electron under certain experimental conditions will be able to accept an electron under different experimental conditions.

It is to be understood that the number of possible transition metal complexes is very large, and that one skilled in the art of electron transfer compounds will be able to utilize a number of compounds in the present invention. By “transitional metal” herein is meant metals whose atoms have a partial or completed shell of electrons. Suitable transition metals for use in the invention include, but are not limited to, cadmium (Cd), copper (Cu), cobalt (Co), palladium (Pd), zinc (Zn), iron (Fe), ruthenium (Ru), rhodium (Rh), osmium (Os), rhenium (Re), platinum (Pt), scandium (Sc), titanium (Ti), Vanadium (V), chromium (Cr), manganese (Mn), nickel (Ni), Molybdenum (Mo), technetium (Tc), tungsten (W), and iridium (Ir). That is, the first series of transition metals, the platinum metals (Ru, Rh, Pd, Os, Ir and Pt), along with Fe, Re, W, Mo and Tc, find particular use in the present invention. Particularly preferred are metals that do not change the number of coordination sites upon a change in oxidation state, including ruthenium, osmium, iron, platinum and palladium, with osmium, ruthenium and iron being especially preferred, and osmium finding particular use in many embodiments. In some embodiments, iron is not preferred. Generally, transition metals are depicted herein as TM or M.

The transitional metal and the coordinating ligands form a metal complex. By “ligand” or “coordinating ligand” (depicted herein in the figures as “L”) herein is meant an atom, ion, molecule, or functional group that generally donates one or more of its electrons through a coordinate covalent bond to, or shares its electrons through a covalent bond with, one or more central atoms or ions.

The other coordination sites of the metal are used for attachment of the transition metal complex to either a capture ligand (directly or indirectly using a linker), or to the electrode (frequently using a spacer, as is more fully described below), or both. Thus for example, when the transition metal complex is directly joined to a binding ligand, one, two or more of the coordination sites of the metal ion may be occupied by coordination atoms supplied by the binding ligand (or by the linker, if indirectly joined). In addition, or alternatively, one or more of the coordination sites of the metal ion may be occupied by a spacer used to attach the transition metal complex to the electrode. For example, when the transition metal complex is attached to the electrode separately from the binding ligand as is more fully described below, all of the coordination sites of the metal (n) except 1 (n−1) may contain polar ligands.

Suitable small polar ligands, generally depicted herein as “L”, fall into two general categories, as is more fully described herein. In one embodiment, the small polar ligands will be effectively irreversibly bound to the metal ion, due to their characteristics as generally poor leaving groups or as good sigma donors, and the identity of the metal. These ligands may be referred to as “substitutionally inert”. Alternatively, as is more fully described below, the small polar ligands may be reversibly bound to the metal ion, such that upon binding of a target analyte, the analyte may provide one or more coordination atoms for the metal, effectively replacing the small polar ligands, due to their good leaving group properties or poor sigma donor properties. These ligands may be referred to as “substitutionally labile”. The ligands preferably form dipoles, since this will contribute to a high solvent reorganization energy.

Some of the structures of transitional metal complexes are shown below:

L are the co-ligands, that provide the coordination atoms for the binding of the metal ion. As will be appreciated by those in the art, the number and nature of the co-ligands will depend on the coordination number of the metal ion. Mono-, di- or polydentate co-ligands may be used at any position. Thus, for example, when the metal has a coordination number of six, the L from the terminus of the conductive oligomer, the L contributed from the nucleic acid, and r, add up to six. Thus, when the metal has a coordination number of six, r may range from zero (when all coordination atoms are provided by the other two ligands) to four, when all the co-ligands are monodentate. Thus generally, r will be from 0 to 8, depending on the coordination number of the metal ion and the choice of the other ligands.

In one embodiment, the metal ion has a coordination number of six and both the ligand attached to the conductive oligomer and the ligand attached to the nucleic acid are at least bidentate; that is, r is preferably zero, one (i.e. the remaining co-ligand is bidentate) or two (two monodentate co-ligands are used). As will be appreciated in the art, the co-ligands can be the same or different. Suitable ligands fall into two categories: ligands which use nitrogen, oxygen, sulfur, carbon or phosphorus atoms (depending on the metal ion) as the coordination atoms (generally referred to in the literature as sigma (σ) donors) and organometallic ligands such as metallocene ligands (generally referred to in the literature as pi (π) donors, and depicted herein as Lm). Suitable nitrogen donating ligands are well known in the art and include, but are not limited to, cyano (C≡N), NH2; NHR; NRR′; pyridine; pyrazine; isonicotinamide; imidazole; bipyridine and substituted derivatives of bipyridine; terpyridine and substituted derivatives; phenanthrolines, particularly 1,10-phenanthroline (abbreviated phen) and substituted derivatives of phenanthrolines such as 4,7-dimethylphenanthroline and dipyridol[3,2-a:2′,3′-c]phenazine (abbreviated dppz); dipyridophenazine; 1,4,5,8,9,12-hexaazatriphenylene (abbreviated hat); 9,10-phenanthrenequinone diimine (abbreviated phi); 1,4,5,8-tetraazaphenanthrene (abbreviated tap); 1,4,8,11-tetra-azacyclotetradecane (abbreviated cyclam) and isocyanide. Substituted derivatives, including fused derivatives, may also be used. In some embodiments, porphyrins and substituted derivatives of the porphyrin family may be used. See for example, Comprehensive Coordination Chemistry, Ed. Wilkinson et al., Pergammon Press, 1987, Chapters 13.2 (pp 73-98), 21.1 (pp. 813-898) and 21.3 (pp 915-957), all of which are hereby expressly incorporated by reference. As will be appreciated in the art, any ligand donor(1)-bridge-donor(2) where donor (1) binds to the metal and donor(2) is available for interaction with the surrounding medium (solvent, protein, etc.) can be used in the present invention, especially if donor(1) and donor(2) are coupled through a pi system, as in cyanos (C is donor(1), N is donor(2), pi system is the CN triple bond). One example is bipyrimidine, which looks much like bipyridine but has N donors on the “back side” for interactions with the medium. Additional co-ligands include, but are not limited to cyanates, isocyanates (—N═C═O), thiocyanates, isonitrile, N2, O2, carbonyl, halides, alkoxyide, thiolates, amides, phosphides, and sulfur containing compound such as sulfino, sulfonyl, sulfoamino, and sulfamoyl.

In some embodiments, multiple cyanos are used as co-ligand to complex with different metals. For example, seven cyanos bind Re(III); eight bind Mo(IV) and W(IV). Thus at Re(III) with 6 or less cyanos and one or more L, or Mo(IV) or W(IV) with 7 or less cyanos and one or more L can be used in the present invention. The EAM with W(IV) system has particular advantages over the others because it is more inert, easier to prepare, more favorable reduction potential. Generally that a larger CN/L ratio will give larger shifts. Suitable sigma donating ligands using carbon, oxygen, sulfur and phosphorus are known in the art. For example, suitable sigma carbon donors are found in Cotton and Wilkinson, Advanced Organic Chemistry, 5th Edition, John Wiley & Sons, 1988, hereby incorporated by reference; see page 38, for example. Similarly, suitable oxygen ligands include crown ethers, water and others known in the art. Phosphines and substituted phosphines are also suitable; see page 38 of Cotton and Wilkinson. The oxygen, sulfur, phosphorus and nitrogen-donating ligands are attached in such a manner as to allow the heteroatoms to serve as coordination atoms.

In some embodiments, organometallic ligands are used. In addition to purely organic compounds for use as redox moieties, and various transition metal coordination complexes with δ-bonded organic ligand with donor atoms as heterocyclic or exocyclic substituents, there is available a wide variety of transition metal organometallic compounds with .pi.-bonded organic ligands (see Advanced Inorganic Chemistry, 5th Ed., Cotton & Wilkinson, John Wiley & Sons, 1988, chapter 26; Organometallics, A Concise Introduction, Elschenbroich et al., 2nd Ed., 1992, VCH; and Comprehensive Organometallic Chemistry II, A Review of the Literature 1982-1994, Abel et al. Ed., Vol. 7, chapters 7, 8, 10 & 11, Pergamon Press, hereby expressly incorporated by reference). Such organometallic ligands include cyclic aromatic compounds such as the cyclopentadienide ion [C5H5 (−1)] and various ring substituted and ring fused derivatives, such as the indenylide (−1) ion, that yield a class of bis(cyclopentadienyl)metal compounds, (i.e. the metallocenes); see for example Robins et al., J. Am. Chem. Soc. 104:1882-1893 (1982); and Gassman et al., J. Am. Chem. Soc. 108:4228-4229 (1986), incorporated by reference. Of these, ferrocene [(C5H5)2 Fe] and its derivatives are prototypical examples which have been used in a wide variety of chemical (Connelly et al., Chem. Rev. 96:877-910 (1996), incorporated by reference) and electrochemical (Geiger et al., Advances in Organometallic Chemistry 23:1-93; and Geiger et al., Advances in Organometallic Chemistry 24:87, incorporated by reference) electron transfer or “redox” reactions. Metallocene derivatives of a variety of the first, second and third row transition metals are potential candidates as redox moieties that are covalently attached to either the ribose ring or the nucleoside base of nucleic acid. Other potentially suitable organometallic ligands include cyclic arenes such as benzene, to yield bis(arene)metal compounds and their ring substituted and ring fused derivatives, of which bis(benzene)chromium is a prototypical example. Other acyclic π-bonded ligands such as the allyl(−1) ion, or butadiene yield potentially suitable organometallic compounds, and all such ligands, in conduction with other .pi.-bonded and .delta.-bonded ligands constitute the general class of organometallic compounds in which there is a metal to carbon bond. Electrochemical studies of various dimers and oligomers of such compounds with bridging organic ligands, and additional non-bridging ligands, as well as with and without metal-metal bonds are potential candidate redox moieties in nucleic acid analysis. When one or more of the co-ligands is an organometallic ligand, the ligand is generally attached via one of the carbon atoms of the organometallic ligand, although attachment may be via other atoms for heterocyclic ligands. Preferred organometallic ligands include metallocene ligands, including substituted derivatives and the metalloceneophanes (see page 1174 of Cotton and Wilkinson, supra). For example, derivatives of metallocene ligands such as methylcyclopentadienyl, with multiple methyl groups being preferred, such as pentamethylcyclopentadienyl, can be used to increase the stability of the metallocene. In a preferred embodiment, only one of the two metallocene ligands of a metallocene are derivatized. As described herein, any combination of ligands may be used. Preferred combinations include: a) all ligands are nitrogen donating ligands; b) all ligands are organometallic ligands; and c) the ligand at the terminus of the conductive oligomer is a metallocene ligand and the ligand provided by the nucleic acid is a nitrogen donating ligand, with the other ligands, if needed, are either nitrogen donating ligands or metallocene ligands, or a mixture. As a general rule, EAM comprising non-macrocyclic chelators are bound to metal ions to form non-macrocyclic chelate compounds, since the presence of the metal allows for multiple proligands to bind together to give multiple oxidation states.

In some embodiments, nitrogen donating proligands are used. Suitable nitrogen donating proligands are well known in the art and include, but are not limited to, NH2; NHR; NRR′; pyridine; pyrazine; isonicotinamide; imidazole; bipyridine and substituted derivatives of bipyridine; terpyridine and substituted derivatives; phenanthrolines, particularly 1,10-phenanthroline (abbreviated phen) and substituted derivatives of phenanthrolines such as 4,7-dimethylphenanthroline and dipyridol[3,2-a:2′,3′-c]phenazine (abbreviated dppz); dipyridophenazine; 1,4,5,8,9,12-hexaazatriphenylene (abbreviated hat); 9,10-phenanthrenequinone diimine (abbreviated phi); 1,4,5,8-tetraazaphenanthrene (abbreviated tap); 1,4,8,11-tetra-azacyclotetradecane (abbreviated cyclam) and isocyanide. Substituted derivatives, including fused derivatives, may also be used. It should be noted that macrocylic ligands that do not coordinatively saturate the metal ion, and which require the addition of another proligand, are considered non-macrocyclic for this purpose. As will be appreciated by those in the art, it is possible to covalent attach a number of “non-macrocyclic” ligands to form a coordinatively saturated compound, but that is lacking a cyclic skeleton.

In some embodiments, a mixture of monodentate (e.g. at least one cyano ligand), bi-dentate, tri-dentate, and polydentate ligands (till to saturate) can be used in the construction of EAMs Generally, it is the composition or characteristics of the ligands that determine whether a transition metal complex is solvent accessible. By “solvent accessible transition metal complex” or grammatical equivalents herein is meant a transition metal complex that has at least one, preferably two, and more preferably three, four or more small polar ligands. The actual number of polar ligands will depend on the coordination number (n) of the metal ion. Preferred numbers of polar ligands are (n−1) and (n-2). For example, for hexacoordinate metals, such as Fe, Ru, and Os, solvent accessible transition metal complexes preferably have one to five small polar ligands, with two to five being preferred, and three to five being particularly preferred, depending on the requirement for the other sites, as is more fully described below. Tetracoordinate metals such as Pt and Pd preferably have one, two or three small polar ligands. It should be understood that “solvent accessible” and “solvent inhibited” are relative terms. That is, at high applied energy, even a solvent accessible transition metal complex may be induced to transfer an electron.

Some examples of EAMs are described herein.

1). Cyano-Based Complexes

In one aspect, the present invention provides EAMs with a transition metal and at least one cyano (—C≡N) ligand. Depending on the valency of the metal and the configuration of the system (e.g. capture ligand contributing a coordination atom, etc.), 1, 2, 3, 4 or 5 cyano ligands can be used. In general, embodiments which use the most cyano ligands are preferred; again, this depends on the configuration of the system. For example, as depicted in FIG. 15, an EAM using a hexadentate metal such as osmium, separately attached from the capture ligand, allows 5 cyano ligands, with the 6th coordination site being occupied by the terminus of the attachment linker. When a hexadentate metal has both an attachment linker and a capture ligand providing coordination atoms, there can be four cyano ligands.

In some embodiments, such as depicted in the Figures, the attachment linker and/or the capture ligand can provide more than a single coordination atom. Thus, for example, in FIG. 17, the attachment linker comprises a bipyridine which contributes two coordination atoms. Compounds 301, 302, 303, 304, 305, and 306 are additional examples of EAMs comprising bipyridine (also denoted bipyridyl) ligands.

In some embodiments, ligands other than cyano ligands are used in combination with at least one cyano ligand.

2). Ru—N Based Complexes

In one aspect, the resent invention provides new architectures for Ru—N based complexes, where the coordination could be monodentate, bidentate, tridentate, or multidendate. Thus the number of coordination ligand L (which covalently connected to the anchor and capture ligand) can be 1, 2, 3, or 4. Some of the examples are shown in FIG. 5A.

The charge-neutralizing ligands can be any suitable ligand known in the art, such as dithiocarbamate, benzenedithiolate, or Schiff base as described herein. The capture ligand and the anchor can be on the same framework or separate.

In another aspect of the present invention, each component of the EAM ligand architecture is connected through covalent bonds rather than Ru coordination chemistry. The construction of the architectures provide herein relies on modern synthetic organic chemical methodology. An important design consideration includes the necessary orthogonal reactivity of the functional groups present in the anchor and capture ligand component versus the coordinating ligand component. Preferably, the entire compound can be synthesized and the redox active transitional metal coordinated to the ligand near the last step of the synthesis. The coordinating ligands provided herein rely on well-established inorganic methodologies for ruthenium pentaamine precursors. See Gerhardt and Weck, J. Org. Chem. 71:6336-6341 (2006); Sizova et al., Inorg. Chim. Acta, 357:354-360 (2004); and Scott and Nolan, Eur. J. Inorg. Chem. 1815-1828 (2005), all herein incorporated by reference. Some examples of EAM architectures with Ru-pentaamine complexes are shown below in FIG. 5B. As can be understood by those skilled in the art, the anchor components of the compounds provided herein could be interchanged between alkyl and multipodal-based thiols.

3). Ferrocene-Based EAMs

In some embodiments, the EAMs comprise substituted ferrocenes. Ferrocene is air-stable. It can be easily substituted with both capture ligand and anchoring group. Upon binding of the target protein to the capture ligand on the ferrocene which will not only change the environment around the ferrocene, but also prevent the cyclopentadienyl rings from spinning, which will change the energy by approximately 4 kJ/mol. WO/1998/57159; Heinze and Schlenker, Eur. J. Inorg. Chem. 2974-2988 (2004); Heinze and Schlenker, Eur. J. Inorg. Chem. 66-71 (2005); and Holleman-Wiberg, Inorganic Chemistry, Academic Press 34th Ed, at 1620, all incorporated by reference.

In some embodiments the anchor and capture ligands are attached to the same ligand for easier synthesis. In some embodiments the anchor and capture ligand are attached to different ligands.

There are many ligands that can be used to build the new architecture disclosed herein. They include but not limited to carboxylate, amine, thiolate, phosphine, imidazole, pyridine, bipyridine, terpyridine, tacn (1,4,7-Triazacyclononane), salen (N,N′-bis(salicylidene) ethylenediamine), acacen (N,N′-Ethylenebis(acetylacetoniminate(−)), EDTA (ethylenediamine tetraacetic acid), DTPA (diethylene triamine pentaacetic acid), Cp (cyclopentadienyl), pincer ligands, and scorpionates. In some embodiments, the preferred ligand is pentaamine.

Pincer ligands are a specific type of chelating ligand. A pincer ligand wraps itself around the metal center to create bonds on opposite sides of the metal as well as one in between. The effects pincer ligand chemistry on the metal core electrons is similar to amines, phosphines, and mixed donor ligands. This creates a unique chemical situation where the activity of the metal can be tailored. For example, since there is such a high demand on the sterics of the complex in order to accommodate a pincer ligand, the reactions that the metal can participate in is limited and selective.

Scorpionate ligand refers to a tridentate ligand which would bind to a metal in a fac manner. The most popular class of scorpionates are the tris(pyrazolyl)hydroborates or Tp ligands. A Cp ligand is isolobal to Tp

In some embodiments, the following restraints are desirable: the metal complex should have small polar ligands that allow close contact with the solvent.

4). Osmium-Based EAMs

In some embodiments, the EAMs comprise substituted osmium complexes defined by the chemical formula R¹,R²-bpy-Os(CN)₄ wherein R¹ and R² are as described herein. The structural formula IV of R¹,R²-bpy-Os(CN)₄ is shown below. The preferred 4,4′ and 5,5′ osmium complex isomers are also shown below as structural formulae IVa and IVb. Osmium complexes appear to be attractive candidates for use in electrochemical biosensor platforms due to their extraordinary redox sensitivity to solvation environment, good optical absorption in the visible region and as strong reductants in both the ground and excited states, relative to ferrocene-based complexes. However, osmium complexes are less air stable, relative to ferrocene complexes. Moreover, while Olabe and co-workers had described preparation and characterization of unsubstituted Os^(II)(bipy)(CN)₄ ²⁻ complexes, unsubstituted osmium complexes however lack sufficient synthetic functionality that would allow them to be incorporated into more elaborate systems. See, for instance, Ward et al. Dalton Trans., 2010, Vol. 39, pp. 8851-8867; Baca et al. Inorg. Chem. 2007, Vol. 46, pp. 9770-9789; and Baca et al., Inorg. Chem, 2008, Vol. 47, pp. 3736-3747. Because osmium complexes can be less chemically stable, air stable, water stable, and/or because prior reported Os^(II)(bpy)(CN)₄ ²⁻ complexes lack sufficient synthetic functionality, the commercial applicability of Os^(II)(bpy)(CN)₄ ²⁻ complexes as probes for biomolecule ligand-receptor interactions have not been explored.

The invention provides the first examples of a new series of mono- and di-amine-functionalized osmium complexes defined by chemical formula R¹,R²-bpy-Os(CN)₄ wherein R¹ and R² are as described herein. Representative osmium complexes of the aforementioned formula corresponding to compounds 301 to 306 are described herein. The synthesis and electrochemical characterization of each complex in solution as described in the Examples demonstrates the surprising and unexpected stability of the Os^(II)/Os^(III) redox couple and the ability to modify the oxidation potential based on pH. Furthermore, the amine functionality can serve as synthetic handles for further functionalization or as basic sites for pH-induced modification of complex properties.

5). Charge-Neutralizing Ligands

In another aspect, the present invention provides compositions having metal complexes comprising charged ligands. The reorganization energy for a system that changes from neutral to charged or from charged to neutral (e.g. M(L)_(n) ⁺<->M(L)_(n) ⁰; M(L)_(n)<->M(L)_(n) ⁰) may be larger than that for a system in which the charge simply changes (e.g. M(L)_(n) ²⁺<->M(L)_(n) ³⁺) because the water molecules and surrounding ions have to “reorganize” more to accommodate the change to or from an uncharged state.

In some embodiments, charged ligand anionic compounds can be used to attach the anchor and the capture ligand to the metal center. A metal complex containing a halide ion X in the inner complex sphere reacts with charged ligands, including but not limited to, thiols (R—SH), thiolates (RS-E; E=leaving group, i.e., trimethylsilyl-group), carbonic acids, dithiols, carbonates, acetylacetonates, salicylates, cysteine, 3-mercapto-2-(mercaptomethyl) propanoic acid. The driving force for this reaction is the formation of HX or EX. If the anionic ligand contains both capture ligand and anchor, one substitution reaction is required, and therefore the metal complex, with which it is reacted, needs to have one halide ligand in the inner sphere. If the anchor and capture ligand are introduced separately the starting material generally needs to contain two halide in the inner coordination sphere. Seidel et al., Inorg. Chem 37:6587-6596 (1998); Kathari and Busch, Inorg. Chem. 8:2276-2280 (1978); Isied and Kuehn J. Am. Chem. Soc. 100:6752-6754; and Volkers et al., Eur. J. Inorg. Chem. 4793-4799 (2006), all herein incorporated by reference.

Examples for suitable metal complexes are the following (it should be noted that the structures depicted below show multiple unidentate ligands, and multidentate ligands can be substituted for or combined with unidentate ligands such as cyano ligands):

In some embodiments, dithiocarbamate is used as a charge-neutralizing ligand, such as the following example:

In some embodiments, benzenedithiolate is used as charge-neutralizing ligand, such as the following example:

In the above depicted structures, Ln is coordinate ligand and n=0 or 1.

In some embodiments, the EAM comprises Schiff base type complexes. By “Schiff base” or “azomethine” herein is meant a functional group that contains a carbon-nitrogen double bond with the nitrogen atom connected to an aryl or alkyl group—but not hydrogen. Schiff bases are of the general formula R₁R₂C═N—R₃, where R₃ is a phenyl or alkyl group that makes the Schiff base a stable imine. Schiff bases can be synthesized from an aromatic amine and a carbonyl compound by nucleophilic addition forming a hemiaminal, followed by a dehydration to generate an imine.

Acacen is a small planar tetradentate ligand that can form hydrogen bonds to surrounding water molecules through its nitrogen and oxygen atoms, which would enhance the reorganization energy effect. It can be modified with many functionalities, including but not limited to, carboxylic acid and halides, which can be used to couple the acacen-ligand to the capture ligand and to the anchoring group. This system allows a large variety of different metal centers to be utilized in the EAMs. Since the ligand binds with its two oxygen and two nitrogen atoms, only four coordination sites are occupied. This leaves two additional coordination sites open, depending on the metal center. These coordination sites can be occupied by a large variety of organic and inorganic ligands. These additional open sites can be used for inner-sphere substitution (e.g. labile H₂O or NH₃ can be displaced by protein binding) or outer-sphere influence (e.g. CO, CN can for H-bonds) to optimize the shift of potentials upon binding of the capture ligand to the target. WO/1998/057158, WO/1997/21431, Louie et al., PNAS 95:6663-6668 (1999), and Bottcher et al., Inorg. Chem. 36:2498-2504 (1997), herein all incorporated by reference.

In some embodiments, salen-complexes are used as well. Syamal et al., Reactive and Functional Polymers 39:27-35 (1999).

The structures of some acacen-based complexes and salen-based complexes are shown below, where positions on the ligand that are suitable for functionalization with the capture ligand and/or the anchor are marked with an asterisk.

One example of using acacen as ligand to form a cobalt complex is the following:

wherein is A and B are substitute groups, Ln is coordinating ligand and n=0 or 1, and Y is a counterion.

6). Sulfato Ligands

In some embodiments, the EAM comprises sulfato complexes, including but not limited to, [L-Ru(III)(NH₃)₄SO₄]⁺ and [L-Ru(III)(NH₃)₄SO2₂]²⁺. The SO₄—Ru(III)-complexes are air stable. The ligand L comprises a capture ligand and anchor. The sulfate ligand is more polar than amine and negatively charged. The surface complexes therefore will have a larger reorganization energy contribution from surrounding water molecules than both the [L-Ru(NH₃)₄-L′] and [L-Ru(NH₃)₅]²⁺. Isied and Taube, Inorg. Chem. 13:1545-1551 (1974), herein incorporated by reference.

7). EAM With Multiple Metals

In one aspect of the present invention multiple metal centers are incorporated to a single ligand complex and thereby increases the signal. This arrangement increases the ratio of metals per target, resulting in a higher sensitivity.

In some embodiments, multiple metal centers are present close to the capture ligand of an anchoring moiety to enable larger interaction with the analyte of interest (target). Having more than one reporter metal per analyte could boost the signal to noise ratio, increasing the sensitivity of the device. One of such examples is shown in FIG. 19.

In some embodiments Prussian blue (PB) is used. Prussian blue is an inorganic, three-dimensional polymer (see below) that can be formed chemically or electrochemically from simple iron cyanide precursors. Other metals such as Mn and V and Ru have also been incorporated into PB-like films. Films of 50-100 nm thickness are formed quickly. An iron (or other similar metal) complex with a capture ligand can be incorporated on the surface of the film by combining this complex with the precursors during the formation of the film:

Fe(H₂O)₆ ³⁺+Fe(CL)(H₂O)₅ ³⁺+Fe(CN)₆ ⁴⁻→PB with Fe—CL on surface

When the target analyte (such as a protein) binds, the hydrogen bonding of water to cyano groups and other water molecules on the surface will be disrupted, and will affect more metals than just the one with the capture ligand. The electrochemical signal will be drastically changed due to this amplification. See FIG. 6A. In some embodiments a background subtraction of the signal before protein binding may be advantageous.

In the example shown above, the axial positions on the iron metal are functionalized with capture ligands arranged orthogonal to the surface. The binding of a single target to the functionalized surface will impact the metal that is directly attached as well as adjacent metal centers. The Fe—CL complex may be added after initial formation of the film so that it will be incorporated on the surface only, and not cause excessive defects. The film thickness can be controlled by the time the chemical reaction is allowed to proceed or by how much current is applied to the solution if formed electrochemically. Alternatively, islands of PB can be grown between areas of alkane or conjugated SAMs with or without the capture ligand. This would require nanopatterning and would help prevent electrochemical signals from extraneous species in the sample solution. The metal can be used include, but not limited to, ruthenium, iron, rhenium, and osmium, with the appropriate ligand structure associated with each. When there are multiple metals in the same complex the connectivity between the multiple metal centers generally should not allow “cross-talk” between the metals; but should rather be insulating.

8). Crown Ether Ligands

In one aspect, the present invention provides compositions where polar groups, such as crown ethers (CEs), are introduced in the vicinity to the metal center. This could increase the potential shift upon binding of a target analyte (e.g. a protein) to an EAM and therefore increase the sensitivity of the probe. Crown ethers are heterocyclic chemical compounds that, in their simplest form, are cyclic oligomers of ethylene oxide. The essential repeating unit of any simple crown ether is ethyleneoxy, i.e., —CH₂CH₂O—, which repeats twice in dioxane and six times in 18-crown-6. In general, macrocycles of the (—CH₂CH₂O—)_(n) type in which n 4 are referred to as “crown” ethers rather than by their lengthier systematic names: for instance, the systematic name of 18-crown-6 is “1,4,7,10,13,16-hexaoxacyclooctadecane.” The first number in a crown ether's name refers to the number of atoms in the cycle, and the second number refers to the number of those atoms which are oxygen. Also envisioned by the present invention are crown ether derivatives. This embodiment of the is based on having a second-sphere moiety bound to the ruthenium center of the EAM prior to the protein binding event. It has been shown that having a crown ether hydrogen bonded to pentaammine ruthenium complexes shifts the redox potential significantly (up to −100 mV) negative in acetonitrile. See Ando, Coordination Chemistry Reviews, 248:185-203 (2004), and references therein; Ando et al., Polyhedron, 11:2335-2340 (1992); Zang et al., Inorg. Chem., 4738-4743 (1994); Todd et al. Inorg. Chem., 32:2001-2001 (1993); Dong et al., J. Am. Chem. Soc., 115, 4379-4380 (1993), all herein incorporated by reference. The applicants have observed a positive shift in redox potential upon protein binding to the EAM provided herein, thus the use of crown ether would amplify this effect. By having some electron donating moiety (i.e. crown ether) bound to the metal center prior to protein binding “stacks the deck” for us by moving our initial potential more negative such that upon protein binding the crown ether is displaced (changing the second sphere coordination) giving a larger positive potential shift. Without being bound by theory, the reason for the increased potential shift is likely the following: CEs form hydrogen bonds to surrounding water molecules. CEs are known to bind to alkali metal ions (e.g. Na⁺, K⁺ in electrolyte) which bind to the oxygen atoms of the CE. In water, the CE, the ion and the counterion (e.g. CD are hydrated with surrounding water molecules. Upon binding of the target to the capture ligand the water molecules surrounding the transition metal complex are replaced as well as the water molecules hydrating the CE, the alkali metal ion and the counterion of the alkali metal ion. See FIGS. 6A and 6B. The fact that many more water molecules are replaced will increase the shift in potential observed in a binding event. In some cases the change in environment from hydrophilic to more hydrophobic actually expels the alkali metal salt (such as K⁺ and Na⁺ ions) from the CE as well as the water molecules, and a potential shift of as much as 1.0V can be expected. Electrochimica Acta 2001, 2733, herein incorporated by reference. See. e.g. FIG. 7. Some of the examples are:

wherein S¹ and S² are spacers, CL=capture ligand and AG=anchoring group.

9). Pyridine-Thioether/Ether-Ligands

In some embodiments, pyridine-thioether/ether-ligands are used in the synthesis of EAM. These ligand systems will be able to bind to various metal centers via the pyridine-nitrogen and the thioether/ether functionality. Without being bound by the theory, there is the possibility that upon binding of the EAM to the target, the metal-thioether/ether bond is getting cleaved and e.g. halide binds to the metal center, which would be an inner-sphere effect, leading to a large shift in the electrochemical potential.

One of the examples for such complexes is shown below:

where Ln=coordinate ligand of metal center, L=0 or 1, and X=O or S;

In some embodiments, bipyridines and other multidentate-nitrogen-based ligands, such as 1,10 phenanthrolines or terpyridines, are used. Examples for these type of ligands are shown below:

D. Spacer Groups

In some embodiments, the EAM or ReAMC is covalently attached to the anchor group (which is attached to the electrode) via an attachment linker or spacer (“Spacer 1”), that further generally includes a functional moiety that allows the association of the attachment linker to the electrode. See for example U.S. Pat. No. 7,384,749, incorporated herein by reference in its entirety and specifically for the discussion of attachment linkers). It should be noted in the case of a gold electrode, a sulfur atom can be used as the functional group (this attachment is considered covalent for the purposes of this invention). By “spacer” or “attachment linker” herein is meant a moiety which holds the redox active complex off the surface of the electrode. In some embodiments, the spacer is a conductive oligomer as outlined herein, although suitable spacer moieties include passivation agents and insulators as outlined below. In some cases, the spacer molecules are SAM forming species. The spacer moieties may be substantially non-conductive, although preferably (but not required) is that the electron coupling between the redox active molecule and the electrode (HAB) does not limit the rate in electron transfer.

In addition, attachment linkers can be used to between the coordination atom of the capture ligand and the capture ligand itself, in the case when ReAMCs are utilized. Similarly, attachment linkers can be branched, such as shown in FIGS. 12-14. In addition, attachment linkers can be used to attach capture ligands to the electrode when they are not associated in a ReAMC. One end of the attachment linker is linked to the EAM/ReAMC/capture ligand, and the other end (although as will be appreciated by those in the art, it need not be the exact terminus for either) is attached to the electrode. The covalent attachment of the conductive oligomer containing the redox active molecule (and the attachment of other spacer molecules) may be accomplished in a variety of ways, depending on the electrode and the conductive oligomer used. See for example Structures 12-19 and the accompanying text in U.S. Patent Publication No. 20020009810, hereby incorporated by reference in its entirety.

In general, the length of the spacer is as outlined for conductive polymers and passivation agents in U.S. Pat. Nos. 6,013,459, 6,013,170, and 6,248,229, as well as U.S. Pat. No. 7,384,749 all herein incorporated by reference in their entireties. As will be appreciated by those in the art, if the spacer becomes too long, the electronic coupling between the redox active molecule and the electrode will decrease rapidly.

E. Capture Ligands

A variety of molecules can be used in the present invention as capture ligands. By “capture ligand” or “binding ligand” or “capture binding ligand” or “capture binding species” or “capture probe” herein is meant a compound that is used to probe for the presence of the target analyte that will bind to the target analyte. Generally, the capture ligand allows the attachment of a target analyte to the electrode, for the purposes of detection. As is more fully outlined below, attachment of the target analyte to the capture probe may be direct (i.e. the target analyte binds to the capture ligand) or indirect (one or more capture extender ligands are used). By “covalently attached” herein is meant that two moieties are attached by at least one bond, including sigma bonds, pi bonds and coordination bonds.

In some embodiments, the binding is specific, and the capture ligand is part of a binding pair. By “specifically bind” herein is meant that the ligand binds the analyte, with specificity sufficient to differentiate between the analyte and other components or contaminants of the test sample. However, as will be appreciated by those in the art, it will be possible to detect analytes using binding which is not highly specific; for example, the systems may use different capture ligands, for example an array of different ligands, and detection of any particular analyte is via its “signature” of binding to a panel of binding ligands, similar to the manner in which “electronic noses” work. This finds particular utility in the detection of chemical analytes. The binding should be sufficient to remain bound under the conditions of the assay, including wash steps to remove non-specific binding. This binding should be sufficient to remain bound under the conditions of the assay, including wash steps to remove non-specific binding. Generally, the disassociation constants of the analyte to the binding ligand will be in the range of at least 10-4-10-6 M−1, with a preferred range being 10-5 to 10-9 M−1 and a particularly preferred range being 10-7-10-9 M−1. As will be appreciated by those in the art, the composition of the capture ligand will depend on the composition of the target analyte. Capture ligands to a wide variety of analytes are known or can be readily found using known techniques. For example, when the analyte is a single-stranded nucleic acid, the capture ligand may be a complementary nucleic acid. Similarly, the analyte may be a nucleic acid binding protein and the capture binding ligand is either single-stranded or double stranded nucleic acid; alternatively, the binding ligand may be a nucleic acid-binding protein when the analyte is a single or double-stranded nucleic acid. When the analyte is a protein, the binding ligands include proteins or small molecules. Preferred binding ligand proteins include peptides. For example, when the analyte is an enzyme, suitable binding ligands include substrates and inhibitors. As will be appreciated by those in the art, any two molecules that will associate may be used, either as an analyte or as the binding ligand. Suitable analyte/binding ligand pairs include, but are not limited to, antibodies/antigens, receptors/ligands, proteins/nucleic acid, enzymes/substrates and/or inhibitors, carbohydrates (including glycoproteins and glycolipids)/lectins, proteins/proteins, proteins/small molecules; and carbohydrates and their binding partners are also suitable analyte-binding ligand pairs. These may be wild-type or derivative sequences. In a preferred embodiment, the binding ligands are portions (particularly the extracellular portions) of cell surface receptors that are known to multimerize, such as the growth hormone receptor, glucose transporters (particularly GLUT 4 receptor), transferrin receptor, epidermal growth factor receptor, low density lipoprotein receptor, high density lipoprotein receptor, epidermal growth factor receptor, leptin receptor, interleukin receptors including IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-11, IL-12, IL-13, IL-15, and IL-17 receptors, human growth hormone receptor, VEGF receptor, PDGF receptor, EPO receptor, TPO receptor, ciliary neurotrophic factor receptor, prolactin receptor, and T-cell receptors. As described herein, the capture ligand can be attached to the coordinating ligand and/or anchor via a covalent bond. The method of attachment of the capture binding ligand will generally be done as is known in the art, and will depend on the composition of the attachment linker and the capture binding ligand. In general, the capture binding ligands are attached to the attachment linker through the use of functional groups on each that can then be used for attachment. Preferred functional groups for attachment are amino groups, carboxy groups, oxo groups and thiol groups. These functional groups can then be attached, either directly or through the use of a linker, sometimes depicted herein as “Z”. Linkers are known in the art; for example, homo- or hetero-bifunctional linkers as are well known (see 1994 Pierce Chemical Company catalog, technical section on cross-linkers, pages 155-200, incorporated herein by reference). Preferred Z linkers include, but are not limited to, alkyl groups (including substituted alkyl groups and alkyl groups containing heteroatom moieties), with short alkyl groups, esters, amide, amine, epoxy groups and ethylene glycol and derivatives being preferred. Z may also be a sulfone group, forming sulfonamide.

In this way, capture binding ligands comprising proteins, lectins, nucleic acids, small organic molecules, carbohydrates, etc. can be added.

In some embodiment, antibodies or a fragment thereof are used as capture ligands. By “antibody” herein is meant a member of a family of glycosylated proteins called immunoglobulins, which can specifically combine with an antigen. The term “antibody” includes full-length as well antibody fragments, as are known in the art, including Fab, Fab2, single chain antibodies (Fv for example), chimeric antibodies, humanized and human antibodies, etc., either produced by the modification of whole antibodies or those synthesized de novo using recombinant DNA technologies, and derivatives thereof. However, in some embodiments, whole antibodies are not preferred. This is because antibodies could be too bulky, leads to interference with transducer. Thus in some embodiments, antibody fragments and mimitopes are used as capture ligands. By “epitope” herein is meant the actual site of antibody recognition of the antigen. The term is also used interchangeably with “antigenic determinant” or “antigenic determinant site”. By “mimitopes” or “mimotope” herein is meant a peptide which has the spatial structure of a biologically important site, e.g., an epitope, or an enzyme active site, or a receptor binding site.

In some embodiments, the capture ligand comprises antibody alternatives, including but not limited to avimer. By “avimer” herein is meant proteins that are evolved from a large family of human extracellular receptor domains by in vitro exon shuffling and phage display. It is generally a multidomain protein with binding and inhibitory properties. See Silverman et al., Nature Biotechnology 23:1556-1561 (2005), herein incorporated by reference.

In some embodiments, the capture ligand comprises oligomeric peptides. These peptides can be obtained using techniques known in the art, including but not limited to phage display, Sidhu et al., Methods Enzymol., 328, 333-363 (2000), and one bead one peptide. For example, the peptide can be obtained using Biopanning. Giodano et al., Nat Med. 7:1249-53 (2001); herein incorporated by reference. The capture ligand may be nucleic acid, when the target analyte is a nucleic acid or nucleic acid binding proteins; alternatively, as is generally described in U.S. Pat. Nos. 5,270,163, 5,475,096, 5,567,588, 5,595,877, 5,637,459, 5,683,867,5,705,337, and related patents, hereby incorporated by reference, nucleic acid “aptamers” can be developed for binding to virtually any target analyte. Similarly, there is a wide body of literature relating to the development of binding partners based on combinatorial chemistry methods. In this embodiment, when the capture ligand is a nucleic acid, preferred compositions and techniques are outlined in PCT US97/20014, hereby incorporated by reference.

In some embodiments, the capture ligand comprises an aptamer. By “aptamer” herein is meant a single-stranded, partially single-stranded, partially double-stranded or double-stranded nucleotide sequence, advantageously a replicatable nucleotide sequence, capable of specifically recognizing a selected non-oligonucleotide molecule or group of molecules by a mechanism other than Watson-Crick base pairing or triplex formation. Aptamers disclosed herein include, without limitation, defined sequence segments and sequences comprising nucleotides, ribonucleotides, deoxyribonucleotides, nucleotide analogs, modified nucleotides and nucleotides comprising backbone modifications, branch points and non-nucleotide residues, groups or bridges. Aptamers of the invention include partially and fully single-stranded and double-stranded nucleotide molecules and sequences, synthetic RNA, DNA and chimeric nucleotides, hybrids, duplexes, heteroduplexes, and any ribonucleotide, deoxyribonucleotide or chimeric counterpart thereof and/or corresponding complementary sequence, promoter or primer-annealing sequence needed to amplify, transcribe or replicate all or part of the aptamer molecule or sequence. Aptamers can specifically bind to soluble, insoluble or immobilized selected molecules (e.g., ligands, receptors and effector molecules). Alternatively, the term “aptamer” includes nucleotides capable of shape-specific recognition of chemically bland surfaces by a mechanism distinctly different from specific binding. Aptamers of the instant invention may be selected to specifically recognize a structural shape or surface feature comprising a chemically bland surface (e.g., a silicon chip or carbon nanostructure) rather than the chemical identity of a selected target molecule (e.g., a ligand or receptor). An aptamer may be a molecule unto itself or a sequence segment comprising a nucleotide molecule or group of molecules, e.g., a defined sequence segment or aptameric sequence comprising a synthetic heteropolymer, multivalent heteropolymeric hybrid structure or aptameric multimolecular device.

IV. Method of Making the Compositions of the Invention

As will be appreciated by those in the art, the compositions can be made using a variety of techniques known in the art. See for example the disclosures of U.S. Pat. Nos. 6,013,459, 6,248,229, 7,018,523, 7,267,939, U.S. patent application Ser. Nos. 09/096,593 and 60/980,733, and U.S. Provisional Application titled “Electrochemical Assay for the Detection of Enzymes” which is hereby incorporated herein by reference.

In one embodiment, the compositions of the invention are made as depicted in FIG. 3. In this embodiment, the electrodes comprising a species including a functional group for the attachment of the capture ligand is used, and after the composition is made, a capture ligand with a complementary functional group is added, resulting in essentially spontaneous addition of the capture ligand to the surface. As will be appreciated by those in the art, there are a wide variety of functional groups/complementary functional groups that can be used. Suitable functional groups include, but are not limited to, maleimide, imidoesters, N-hydroxysuccinimidyls, alkyl halides, aryl halides, alpha-haloacyls and pryidyl disulfides. In general, the corresponding/complementary functional groups sulfhydryls, amines, amines, sulfhydryls, sulfhydryls, sulfhydryls and sulfhydryls, respectively. As will be appreciated by those in the art, it is also possible to switch the orientation of these functional groups, e.g. the sulfhydryl is present on the attachment linker and the maleimide is added to the biomolecule to be used as the capture ligand. As noted herein, the methods of attaching are dependent upon the reactive groups present on the two components. In an exemplary embodiment, the reactive functional group of the haptens of the invention and the functional group of the reactive part comprise electrophiles and nucleophiles that can generate a covalent linkage between them. Alternatively, the reactive functional group comprises a photoactivatable group, which becomes chemically reactive only after illumination with light of an appropriate wavelength. Typically, the conjugation reaction between the reactive functional group and the reactive partner results in one or more atoms of the reactive functional group or the reactive partner being incorporated into a new linkage attaching the two components. Selected examples of functional groups and linkages are shown in Table 1, where the reaction of an electrophilic group and a nucleophilic group yields a covalent linkage.

TABLE 1 Examples of some routes to useful covalent linkages with electrophile and nucleophile reactive groups Electrophilic Group Nucleophilic Group Resulting Covalent Linkage activated esters* amines/anilines carboxamides acyl azides** amines/anilines carboxamides acyl halides amines/anilines carboxamides acyl halides alcohols/phenols esters acyl nitriles alcohols/phenols esters acyl nitriles amines/anilines carboxamides aldehydes amines/anilines imines aldehydes or ketones hydrazines hydrazones aldehydes or ketones hydroxylamines oximes alkyl halides amines/anilines alkyl amines alkyl halides carboxylic acids esters alkyl halides thiols thioethers alkyl halides alcohols/phenols ethers alkyl sulfonates thiols thioethers alkyl sulfonates carboxylic acids esters alkyl sulfonates alcohols/phenols ethers anhydrides alcohols/phenols esters anhydrides amines/anilines carboxamides aryl halides thiols thiophenols aryl halides amines aryl amines aziridines thiols thioethers boronates glycols boronate esters carboxylic acids amines/anilines carboxam ides carboxylic acids alcohols esters carboxylic acids hydrazines hydrazides carbodiimides carboxylic acids N-acylureas or anhydrides diazoalkanes carboxylic acids esters epoxides thiols thioethers haloacetamides thiols thioethers halotriazines amines/anilines aminotriazines halotriazines alcohols/phenols triazinyl ethers imido esters amines/anilines amidines isocyanates amines/anilines ureas isocyanates alcohols/phenols urethanes isothiocyanates amines/anilines thioureas maleimides thiols thioethers phosphoramidites alcohols phosphite esters silyl halides alcohols silyl ethers sulfonate esters amines/anilines alkyl amines sulfonate esters thiols thioethers sulfonate esters carboxylic acids esters sulfonate esters alcohols ethers sulfonyl halides amines/anilines sulfonamides sulfonyl halides phenols/alcohols sulfonate esters *Activated esters, as understood in the art, generally have the formula—COΩ, where Ω is a good leaving group (e.g. oxysuccinimidyl (—OC₄H₄O₂) oxysulfosuccinimidyl (—OC₄H₃O₂—SO₃H),-1-oxybenzotriazolyl (—OC₆H₄N₃); or an aryloxy group or aryloxy substituted one or more times by electron withdrawing substituents such as nitro, fluoro, chloro, cyano, or trifluoromethyl, or combinations thereof, used to form activated aryl esters; or a carboxylic acid activated by a carbodiimide to form an anhydride or mixed anhydride—OCOR^(a) or —OCNR^(a)NHR^(b), where R^(a) and R^(b), which may be the same or different, are C₁-C₆ alkyl, C₁-C₆ perfluoroalkyl, or C₁-C₆ alkoxy; or cyclohexyl, 3-dimethylaminopropyl, or N-morpholinoethyl). **Acyl azides can also rearrange to isocyanates

The functional groups and complementary functional groups can also include linkers, for flexibility or steric rigidity as the case may be, or other reasons.

It should be noted that while the figures depict the presence of a functional group and the complementary functional group, in some cases the addition results in the loss of atoms from these groups, and thus this is not meant to depict a situation when the entire functional group and complementary functional group is present in the final composition.

In addition, the figures depict the use of “monofunctional” linkers, e.g. a maleimide. It is also possible to include additional steps that utilize either homo- or heterobifunctional groups, (see 1994 Pierce Chemical Company catalog, technical section on cross linkers, pages 155-200, incorporated herein by reference). For example, an attachment linker comprising a sulfur atom on one terminus and an amino group on the other end could be reacted with a bifunctional linker that reacts with amines, and then subsequently a capture ligand comprising an amino group can be added.

In another embodiment, the compositions of the invention are made by synthesizing each component and adding them to the electrode, generally simultaneously. That is, in the embodiment of FIG. 12A, for example, the REAMC comprising the attachment linker (with the attachment functional moiety such as a sulfur atom), the ligands, the transition metal and the binding ligand is made, and added (optionally with a SAM forming species) to the electrode. Similarly, a two or three component system is done in FIG. 1B, with a first species comprising the EAM with the attachment linker and attachment functional group, a second species comprising an attachment linker with the capture ligand, and the optional third species of a SAM forming species, which are added, against generally simultaneously, to the electrode. In some cases, the components can be added sequentially, and in some cases, a post synthesis step done of adding extra SAM forming species (and/or other components) with optional heating can be done to ensure good packing on the electrode.

In some embodiments, the ligands can have functionalities that allow the anchor and capture ligand to be added to it after the metal complex is formed.

In some embodiments, the compound is synthesized stepwise. Thus, the capture ligand and the anchor are added to the ligand of the EAM sequentially.

In some embodiments, the capture ligand and the anchor are added to the EAM concomitantly.

In some embodiments, “clip” are added to the EAM first and the capture ligand and anchor groups are added to the “clips” later. By “clips” herein is meant a group or moiety that can be attached to an EAM covalently, and on to which the capture ligand and/or the anchor group could be added.

One of the clips is shown below:

wherein PG=protection group.

In some embodiments, wherein pentaammine is used as coordinating ligands, the capture ligand can be added first, and the anchor group is added to the capture ligand. The compositions of the present invention may be used in a variety of research, clinical, quality control, or field testing settings. The examples provided herein are for illustration purposes only and are in no means to limit the scope the present invention. Further, all references cited herein are incorporated by reference for all the relevant contents therein.

V. Method of Using the Composition of the Invention A. Target Analyte and Sample

In one aspect, the present invention provides methods and compositions useful in the detection of target analytes. By “target analyte” or “analyte” or grammatical equivalents herein is meant any molecule or compound to be detected and that can bind to a binding species, e.g. a capture ligand, defined below. Suitable analytes include, but not limited to, small chemical molecules such as environmental or clinical chemical or pollutant or biomolecule, including, but not limited to, pesticides, insecticides, toxins, therapeutic and abused drugs, hormones, antibiotics, antibodies, organic materials, etc. Suitable biomolecules include, but are not limited to, proteins (including enzymes, immunoglobulins and glycoproteins), nucleic acids, lipids, lectins, carbohydrates, hormones, whole cells (including procaryotic (such as pathogenic bacteria) and eucaryotic cells, including mammalian tumor cells), viruses, spores, etc. Particularly preferred analytes are proteins including enzymes; drugs, cells; antibodies; antigens; cellular membrane antigens and receptors (neural, hormonal, nutrient, and cell surface receptors) or their ligands.

In some embodiments, the target analyte is cytochrome P450, avidin/streptavdin, SEB, PSA- (protease), tryprin/chymotrypin (protease), anthrax spore and E. col. 0157:H7.

In some embodiments, the target analyte is a protein. As will be appreciated by those in the art, there are a large number of possible proteinaceous target analytes that may be detected using the present invention. By “proteins” or grammatical equivalents herein is meant proteins, oligopeptides and peptides, derivatives and analogs, including proteins containing non-naturally occurring amino acids and amino acid analogs, and peptidomimetic structures. The side chains may be in either the (R) or the (S) configuration. In a preferred embodiment, the amino acids are in the (S) or L-configuration. As discussed below, when the protein is used as a capture ligand, it may be desirable to utilize protein analogs to retard degradation by sample contaminants. Suitable protein target analytes include, but are not limited to, (1) immunoglobulins, particularly IgEs, IgGs and IgMs, and particularly therapeutically or diagnostically relevant antibodies, including but not limited to, for example, antibodies to human albumin, apolipoproteins (including apolipoprotein E), human chorionic gonadotropin, cortisol, α-fetoprotein, thyroxin, thyroid stimulating hormone (TSH), antithrombin, antibodies to pharmaceuticals (including antieptileptic drugs (phenytoin, primidone, carbariezepin, ethosuximide, valproic acid, and phenobarbitol), cardioactive drugs (digoxin, lidocaine, procainamide, and disopyramide), bronchodilators (theophylline), antibiotics (chloramphenicol, sulfonamides), antidepressants, immunosuppresants, abused drugs (amphetamine, methamphetamine, cannabinoids, cocaine and opiates) and antibodies to any number of viruses (including orthomyxoviruses, (e.g. influenza virus), paramyxoviruses (e.g respiratory syncytial virus, mumps virus, measles virus), adenoviruses, rhinoviruses, coronaviruses, reoviruses, togaviruses (e.g. rubella virus), parvoviruses, poxviruses (e.g. variola virus, vaccinia virus), enteroviruses (e.g. poliovirus, coxsackievirus), hepatitis viruses (including A, B and C), herpesviruses (e.g. Herpes simplex virus, varicella-zoster virus, cytomegalovirus, Epstein-Barr virus), rotaviruses, Norwalk viruses, hantavirus, arenavirus, rhabdovirus (e.g. rabies virus), retroviruses (including HIV, HTLV-I and -II), papovaviruses (e.g. papillomavirus), polyomaviruses, and picornaviruses, and the like), and bacteria (including a wide variety of pathogenic and non-pathogenic prokaryotes of interest including Bacillus; Vibrio, e.g. V. cholerae; Escherichia, e.g. Enterotoxigenic E. coli, Shigella, e.g. S. dysenteriae; Salmonella, e.g. S. typhi; Mycobacterium e.g. M. tuberculosis, M. leprae; Clostridium, e.g. C. botulinum, C. tetani, C. difficile, C. perfringens; Cornyebacterium, e.g. C. diphtheriae; Streptococcus, S. pyogenes, S. pneumoniae; Staphylococcus, e.g. S. aureus; Haemophilus, e.g. H. influenzae; Neisseria, e.g. N. meningitidis, N. gonorrhoeae; Yersinia, e.g. G. lamblia Y. pestis, Pseudomonas, e.g. P. aeruginosa, P. putida; Chlamydia, e.g. C. trachomatis; Bordetella, e.g. B. pertussis; Treponema, e.g. T. palladium; and the like); (2) enzymes (and other proteins), including but not limited to, enzymes used as indicators of or treatment for heart disease, including creatine kinase, lactate dehydrogenase, aspartate amino transferase, troponin T, myoglobin, fibrinogen, cholesterol, triglycerides, thrombin, tissue plasminogen activator (tPA); pancreatic disease indicators including amylase, lipase, chymotrypsin and trypsin; liver function enzymes and proteins including cholinesterase, bilirubin, and alkaline phosphotase; aldolase, prostatic acid phosphatase, terminal deoxynucleotidyl transferase, and bacterial and viral enzymes such as HIV protease; (3) hormones and cytokines (many of which serve as ligands for cellular receptors) such as erythropoietin (EPO), thrombopoietin (TPO), the interleukins (including IL-1 through IL-17), insulin, insulin-like growth factors (including IGF-1 and -2), epidermal growth factor (EGF), transforming 

1. A compound of chemical formula R¹,R²-bpy-Os(CN)₄

wherein R¹ and R² may be the same or different and are chosen from the group consisting of CH₃, CH₃(CH₂)_(m), and NH₂(CH₂)_(n) wherein m and n are independently chosen from the group consisting of 0, 1, 2, 3, 4, 5, and
 6. 2-3. (canceled)
 4. A compound of formula R¹,R²-bpy-Os(CN)₄

wherein R¹ and R² are independently substituted alkyl groups wherein the substituents are chosen from the group consisting of an electrophile and a nucleophile. 5-25. (canceled)
 26. A method of detecting a target enzyme comprising: a) contacting a sample with a composition comprising an electrode comprising: i) an electroactive moiety (EAM) comprising a compound of claim 1 with a first E⁰; ii) a capture ligand; under conditions whereby said target enzyme, if present, alters said capture ligand such that said EAM has a second E⁰; and b) measuring said second E⁰.
 27. The method according to claim 26, wherein said composition comprises a support comprising a plurality of electrodes each comprising: i) an electroactive moiety (EAM) comprising a compound of claim 1 with a first E⁰⁴⁾; ii) a capture ligand.
 28. A method of detecting a target enzyme comprising: a) contacting a sample with a composition comprising a REAMC comprising: i) an electroactive moiety (EAM) comprising a compound of claim 1 with a first E⁰; ii) a capture ligand; under conditions whereby said target enzyme, if present, alters said capture ligand such that said EAM has a second E⁰; and b) measuring said second E⁰.
 29. A composition comprising an electrode comprising: a) a first species comprising a functional group; and b) an electroactive complex (EAM), said EAM comprising a compound of claim
 1. 30. The composition according to claim 29, wherein said functional group is a maleimide group.
 31. A method of making a biosensor comprising: a) providing an electrode comprising: i) a first species comprising a first functional group; and ii) an electroactive complex (EAM), said EAM comprising a compound of claim 1; c) contacting said electrode with a biomolecule comprising a second functional group to form a covalent bond between said first species and said biomolecule.
 32. The method according to claim 31, wherein said first functional group is selected from the group consisting of moieties comprising a maleimide, imidoester, N-hydroxysuccinimidyl, alkyl halide, aryl halide, alpha-haloacyl and pryidyl disulfide.
 33. The method according to claim 31, wherein said first functional group comprises a maleimide and said biomolecule is a protein comprising a cysteine amino acid.
 34. The method according to claim 33, wherein said protein is a peptide.
 35. A compound having the formula: Anchor-Spacer 1-EAM-(Spacer 2)_(n)-CL  (I) wherein said anchor comprises a cyclic-disulfide group, EAM is a compound of claim 1, CL is a capture ligand, Spacer 1 is a SAM forming species, and n=0 or
 1. 36. The compound according to claim 35, wherein said cyclic-disulfide group comprises pyridyl-oligophenylethylnyl-[1,2,5]-dithiazepane.
 37. A composition comprising an electrode comprising a self-assembled monolayer (SAM), wherein said SAM comprises a compound having the formula: Anchor-Spacer 1-EAM-Spacer 2-CL  (II), wherein said anchor is linked to said electrode group through a disulfide group, EAM is a compound of claim 1, CL is a capture ligand, Spacer 1 is either insulating or conducting, and Spacer 2 is optional.
 38. A method, comprising: a) providing a compound having the formula: Anchor-Spacer 1-EAM-(Spacer 2)_(n)-CL  (I), wherein said anchor comprises a cyclic-disulfide group, EAM is a compound of claim 1, CL is a capture ligand, Spacer 1 is either insulating or conduction, and n=0 or 1; and contacting said compound to an electrode by opening said cyclic disulfide to form an attachment of said anchor to said electrode.
 39. The method according to claim 38, wherein said electrode further comprises a self-assembled monolayer (SAM).
 40. A method of detecting a target analyte in a test sample, comprising: b) providing an electrode comprises a compound having the formula: Anchor-Spacer 1-EAM-(Spacer 2)_(n)-CL  (I), wherein said anchor comprises a cyclic-disulfide group, EAM is a compound of claim 1, CL is a capture ligand, Spacer 1 is either insulating or conduction, and n=0 or 1; and contacting said electrode with said test sample; and determining the presence of said target analyte by measuring the reorganization energy of said EAM. 